U.S. patent application number 14/788896 was filed with the patent office on 2015-12-31 for amyloid beta1-6 antigen arrays.
This patent application is currently assigned to Novartis AG. The applicant listed for this patent is Martin F. Bachmann, Peter Frey, Rainer Martin Lueoend, Rainer Ortmann, Matthias Staufenbiel, Alain Tissot. Invention is credited to Martin F. Bachmann, Peter Frey, Rainer Martin Lueoend, Rainer Ortmann, Matthias Staufenbiel, Alain Tissot.
Application Number | 20150374807 14/788896 |
Document ID | / |
Family ID | 31891336 |
Filed Date | 2015-12-31 |
United States Patent
Application |
20150374807 |
Kind Code |
A1 |
Bachmann; Martin F. ; et
al. |
December 31, 2015 |
Amyloid beta1-6 Antigen Arrays
Abstract
The present invention is related to the fields of molecular
biology, virology, immunology and medicine. The invention provides
a composition comprising an ordered and repetitive antigen or
antigenic determinant array, and in particular an
A.beta.1-peptide-VLP-composition. More specifically, the invention
provides a composition comprising a virus-like particle and at
least one A.beta.1-6 peptide bound thereto. The invention also
provides a process for producing the conjugates and the ordered and
repetitive arrays, respectively. The compositions of the invention
are useful in the production of vaccines for the treatment of
Alzheimer's disease and as a pharmaccine to prevent or cure
Alzheimer's disease and to efficiently induce immune responses, in
particular antibody responses. Furthermore, the compositions of the
invention are particularly useful to efficiently induce
self-specific immune, responses within the indicated context.
Inventors: |
Bachmann; Martin F.;
(Seuzach, CH) ; Tissot; Alain; (Zurich, CH)
; Ortmann; Rainer; (Saint Louis, FR) ; Lueoend;
Rainer Martin; (Therwil, CH) ; Staufenbiel;
Matthias; (Lorrach, DE) ; Frey; Peter; (Bern,
CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bachmann; Martin F.
Tissot; Alain
Ortmann; Rainer
Lueoend; Rainer Martin
Staufenbiel; Matthias
Frey; Peter |
Seuzach
Zurich
Saint Louis
Therwil
Lorrach
Bern |
|
CH
CH
FR
CH
DE
CH |
|
|
Assignee: |
Novartis AG
Basel
CH
|
Family ID: |
31891336 |
Appl. No.: |
14/788896 |
Filed: |
July 1, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14105701 |
Dec 13, 2013 |
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14788896 |
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11905986 |
Oct 5, 2007 |
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14105701 |
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10622087 |
Jul 18, 2003 |
7279165 |
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11905986 |
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60470432 |
May 15, 2003 |
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60396639 |
Jul 19, 2002 |
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Current U.S.
Class: |
424/185.1 |
Current CPC
Class: |
A61K 2039/64 20130101;
A61K 2039/6075 20130101; A61K 39/0007 20130101; A61K 2039/5256
20130101; A61P 25/28 20180101; A61P 37/02 20180101; A61P 25/00
20180101; A61K 2039/5258 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00 |
Claims
1. An immunogenic composition comprising: (a) a core particle with
at least one first attachment site, wherein said core particle is a
Virus-like particle of an RNA bacteriophage; and (b) at least one
antigen or antigenic determinant with at least one second
attachment site, wherein said antigen or antigenic determinant is a
A.beta.1-6 peptide, and wherein said second attachment site being
selected from the group consisting of: (i) an attachment site not
naturally occurring with said antigen or antigenic determinant; and
(ii) an attachment site naturally occurring with said antigen or
antigenic determinant, wherein said second attachment site is
capable of association to said first attachment site; and wherein
said A.beta.1-6 peptide and said core particle interact through
said association to form an ordered and repetitive antigen array,
and further wherein said A.beta.1-6 peptide has an amino acid
sequence variant of SEQ ID NO:75 (DAEFRH), wherein the amino acid
at position 1 of the SEQ ID NO: 75 variant is aspartic acid,
alanine or tyrosine, the amino acid at position 2 of the SEQ ID NO:
75 variant is alanine, serine or tyrosine, the amino acid at
position 3 of the SEQ ID NO: 75 variant is glutamic acid, the amino
acid at position 4 of the SEQ ID NO: 75 variant is phenylalanine or
tyrosine, the amino acid at position 5 of the SEQ ID NO: 75 variant
is arginine or glycine, and the amino acid at position 6 of the SEQ
ID NO: 75 variant is histidine, provided that the full-length
sequence of said A.beta.1-6 peptide is not DAEFRH.
2. The immunogenic composition according to claim 1, wherein the
amino acid sequence variant of SEQ ID NO: 75 is selected from the
group consisting of: SEQ ID NO: 76 (DAEFGH), SEQ ID NO: 86
(ASEYRH), and SEQ ID NO: 90 (YYEFRH).
3. The immunogenic composition of claim 1, further comprising an
adjuvant.
4. The immunogenic composition of claim 1, wherein said immunogenic
composition is devoid of an adjuvant.
5. The immunogenic composition of claim 1, wherein said second
attachment site is capable of association to said first attachment
site through at least one covalent bond.
6. The immunogenic composition of claim 1, wherein said composition
further comprising a heterobifunctional cross-linker, which
contains a functional group which can react with said first
attachment site and a further functional group which can react with
said second attachment site.
7. The immunogenic composition of claim 6, wherein said
heterobifunctional cross-linker is selected from the group
consisting of SMPH, Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS Sulfo-SIAB,
Sulfo-SMPB, Sulfo-SMCC, SVSB, and SLA.
8. A pharmaceutical composition comprising: (a) the immunogenic
composition of claim 1; and (b) an acceptable pharmaceutical
carrier.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention is related to the fields of molecular
biology, virology, immunology and medicine. The invention provides
a composition comprising an ordered and repetitive antigen or
antigenic determinant array, and in particular an A.beta.1-6
peptide-VLP-composition. More specifically, the invention provides
a composition comprising a virus-like particle and at least one
A.beta.1-6 peptide bound thereto. The invention also provides a
process for producing the conjugates and the ordered and repetitive
arrays, respectively. The compositions of the invention are useful
in the production of vaccines for the treatment of Alzheimer's
disease and as a pharmaccine to prevent or cure Alzheimer's disease
and to efficiently induce immune responses, in particular antibody
responses. Furthermore, the compositions of the invention are
particularly useful to efficiently induce self-specific immune
responses within the indicated context.
[0003] 2. Related Art
[0004] Alzheimer's disease (AD) is the most common cause of
dementia among the elderly (age 65 and older) and a serious burden
for public health. For example, 4 million people are reported to
suffer from the disease in the United Sates of America. The
incidence of the disease is expected to increase as the population
ages.
[0005] The main pathological signs of Alzheimer's disease are
age-related changes in behaviour, deposition of .beta.-amyloid into
insoluble plaques, called the neuritic plaques or AD plaques,
neurofibrillary tangles composed of tan protein within neurons, and
loss of neurons throughout the forebrain. In addition to the late
onset AD, which occurs in did age (65 years and more), there is an
early onset AD, familial AD (FAD) occurring between age 35 and 60.
The pathological abnormalities of AD are more widespread, severe
and occur earlier in FAD than in late onset or sporadic AD.
Mutations in the APP gene, the presenilin 1 and the presenilin 2
genes have been correlated with FAD.
[0006] As indicated, one of the key events in Alzheimer's Disease
(AD) is the deposition of amyloid as insoluble fibrous masses
(amyloidogenesis) resulting in extracellular neuritic plaques and
deposits around the walls of cerebral blood vessels (for review see
Selkoe, D. J. (1999) Nature, 399, A23-31). The major constituent of
the neuritic plaques and congophilic angiopathy is amyloid .beta.
(A.beta.), although these deposits also contain other proteins such
as glycosaminoglycans and apolipoproteins. A.beta. is
proteolytically cleaved from a much larger glycoprotein known as
Amyloid Precursor Protein (APP), which comprises isoforms of
695-770 amino acids with a single hydrophobic transmembrane region.
A.beta. forms a group of peptides up to 43 amino acids in length
showing considerable amino- and carboxy-terminal heterogeneity
(truncation) as well as modifications (Roher, A. E., Palmer, K. C.,
Chan, V., & Ball, M. J. (1988) J. Cell Biol. 107, 2703-2716.
Roher, A. E., Palmer, K. C., Yurewicz, E. C., Ball M. J., &
Greenberg, B. D. (1993) J. Neurochem. 61, 1916-1926). Prominent
isoforms are A.beta.1-40 and 1-42. It has a high propensity to form
.beta.-sheets aggregating into fibrils, which ultimately leads to
the amyloid.
[0007] A.beta. peptide has a central role in the neuropathology of
Alzheimers disease. Region specific, extracellular accumulation of
A.beta. peptide is accompanied by microgliosis, cytoskeletal
changes, dystrophic neuritis and synaptic loss. These pathological
alterations are thought to be linked to the cognitive decline that
defines the disease.
[0008] Administration of amyloid beta protein or, in particular,
A.beta. 1-28 in amounts of up to 10.sup.-2 mg/dose in the absence
of any adjuvants and without any linkage of the amyloid beta
protein or A.beta. 1-28 to a carrier, for the treatment of
Alzheimer's disease, is described in EP 526,511.
[0009] Others have used administration of A.beta. peptides in
combination with adjuvants, to induce an immune response, cellular
or humoral against A.beta. 1-42. In a transgenic mouse model of
Alzheimer disease, animals overexpress human amyloid precursor
protein containing the mutation APP(717)V-F (PDAPP-mice;
Johnson-Wood, K. et al., Proc. Natl. Acad. USA 94: 1550-1555,
Games, D. et al., Nature 373: 523-527 (1995a)), leading to
overproduction of A.beta..sub.1-42, develop plaques, dystrophic
neuritis, loss of presynaptic terminals, astrocytosis and
microgliosis. In a recent study, Schenk, D. et al., (Nature
400:173-77 (1999) and WO 99/27944) report that administration of
aggregated A.beta..sub.1-42 mixed with a strong adjuvant (CFA/IFA),
which cannot be used in humans, in the first 4 immunizations,
followed by administration of aggregated A.beta.1-42 in PBS in the
subsequent immunizations, to PDAPP-mice at 6 weeks of age,
essentially prevented plaque formation and associated dystrophic
neuritis. The same authors reported that immunization of older mice
(11 months of age) using the same strategy markedly reduced the
extent and progression of Alzheimer's disease (AD)-like
neuropathologies. Proliferation of splenocytes from mice immunized
using the abovementioned strategy was reported in Example III
(Screen for therapeutic Efficacy against established AD) of WO
99/27944, showing that A.beta.1-42 specific T-cells were induced by
the vaccination procedure. Coupling of A.beta. fragments to sheep
anti-mouse IgG, and immunization of said coupled fragment in the
presence of the adjuvant CFA/IFA is reported in WO 9927944. The use
of compositions comprising A.beta. fragments linked to polypeptides
such as diphtheria toxin for promoting art immune response against
A.beta. is also disclosed in WO 99/27944. However, no data of
immunization are provided.
[0010] A monoclonal antibody recognizing an epitope within the
N-terminus (1-16) of A.beta. (antibody 6C6) has been shown to
protect PC12 cells from neurotoxicity of fibrillar .beta.-amyloid,
and to disaggregate .beta.-amyloid in vitro (Solomon B. et al.,
Proc. Natl. Acad. Sci. USA (1997)). A monoclonal antibody raised
against A.beta.1-28, was also shown to suppress .beta.-amyloid
aggregation in vitro (Solomon B. et al., Proc. Natl. Acad. Sci. USA
(1996)). Frenkel et al., (J. Neuroimmunol. 88: 85-90 (1998)) have
later identified the epitope of two anti-aggregating antibodies,
10D5 and 6C6, as being the epitope "EFRH", i.e. A.beta.3-6. In
contrast, an antibody specific for A.beta.1-7 was unable to prevent
.beta.-amyloid aggregation (Frenkel D. et al., J. Neuroimmunol. 95:
136-142 (1999)).
[0011] A.beta.1-42 is fibrillogenic, and indeed, the vaccine
composition described in WO 99/27944 used A.beta.1-42 treated in
such a way that it can form aggregates. It has been shown that
those fibrils are toxic for neuronal cell cultures (Yankner et al.,
Science 243: 417-420 (1989)), and that a toxic effect is also
observed when injected into animal brains (Sigurdson et al.,
Neurobiol. Aging 17: 893-901 (1996); Sigurdson et al., Neurobiol.
Aging 18: 591-608 (1997)). Walsh et al., (Nature 416: 535-539
(2002)) report that natural oligomers of A.beta. are formed within
intracellular vesicles. Those oligomers inhibited long term
potentiation in rats in vivo and disrupted synaptic plasticity at
concentrations found in human brain and cerebrospinal fluid.
[0012] In another study, Bard, F. et al. (Nature Medicine 6: 916-19
(2000)) reported that peripheral administration of antibodies
raised against A.beta..sub.1-42, was able to reduce amyloid burden,
despite relatively modest serum levels. This study utilized either
polyclonal antibodies raised against A.beta..sub.1-42, or
monoclonal antibodies raised against synthetic fragments derived
from different regions of A.beta.. Thus induction of antibodies
against A.beta. peptides bears promises as a potential therapeutic
treatment for Alzheimer disease.
[0013] Mucosal administration of an antigen associated with
.beta.-amyloid plaques, such as .beta.-amyloid peptide and
A.beta.1-40, has been described in WO99/27949. Mucosal
administration is said to suppress certain cytokine responses
associated with Alzheimer's disease, and to enhance certain other
cytokine responses associated with the suppression of inflammatory
responses linked to the disease. It is thought that suppression of
the inflammatory responses is effected by the "elicitation of
T-cells characterized by an anti-inflammatory cytokine profile".
Suitable antigens, as described in WO9927949, include antigens
specific for AD, and which are recognized by immune T-cells of a
human or animal host.
[0014] Fusion of epitopes of a monoclonal antibody recognizing
A.beta. to coat proteins of filamentous phages is described in WO
01/18169. Immunization of mice with the filamentous phages
displaying the 15-mer epitope VHEPHEFRHVALNPV (SEQ ID NO: 89) on
the coat protein VIII resulted in antibodies recognizing A.beta.
1-16, and A.beta.1-40. This was demonstrated in an inhibition ELISA
using A.beta. peptides, and an IC50 of 1 .mu.M was found for
inhibition of the binding of the sera to A.beta.1-16 with
A.beta.1-40. Solomon (WO 01/18169), however, provides no indication
that the sera elicited against the filamentous phages carrying the
VHEPHEFRHVALNPV epitope (SEQ ID NO: 89), bind to amyloid plaques or
neuritic plaques of AD.
[0015] There are a number of drawbacks in using sequences differing
from the antigen against which an immune response is to be elicited
for immunization. First, antibodies against part of the sequence
foreign to the antigen of antigenic determinant may be induced.
Second, the conformation of the antigen in the context of the
foreign flanking sequence element may be different than in the
context of the full-length antigen. Thus, although antibodies
cross-reacting to the antigen may be elicited, their binding to the
antigen may be suboptimal. The fine specificity of those elicited
antibody may also not correspond to the specificity of antibodies
elicited against the antigen itself, as additional sid-chains
different from the residues present on the full-length, A.beta. are
present in the epitope. Finally, a 15-mer amino-acid sequence may
contain T-cell epitopes. Display of the epitope YYEFRH (SEQ ID NO:
90) on the protein III of filamentous phage coat, of which 3-5
copies only are usually present on each phage, is also disclosed in
WO 01/18169. Several problems arise when using infectious phages as
carrier for immunization. First, production of infectious agents in
large scale and in sufficient quantity for large immunization
campaigns is problematic. Second, the presence of the DNA of the
phage containing antibiotic resistance genes in the vaccine is not
unproblematic and is a safety issue. Finally, the feasibility and
efficacy of irradiation of large quantities of phages, in the case
where non-infectious phages are used as vaccine, is unresolved.
[0016] A.beta. analogues, wherein A.beta. is modified to include T
helper epitopes have been described (WO 01/62284). Immunization of
TgRND8+ mice, transgenic for human APP, with the A.beta. analogue
resulted in a 4- to 7.5-fold higher antibody titer over
immunization with A.beta.1-42 in the absence of adjuvant.
[0017] Recent studies demonstrated that a vaccination-induced
reduction in brain amyloid deposits has the potential to improve
cognitive functions (Schenk, D., et al. Nature 400: 173-177 (1999);
Janus, C. et al., Nature 408: 979-982 (2000); Morgan, D. et al.,
Nature 408: 982-985 (2000)).
[0018] The autopsy of a patient immunised with aggregated
A.beta.1-42 in the Adjuvant QS21 has revealed the presence of a
T-lymphocyte meningoencephalitis and infiltration of cerebral white
matter by macrophages (Nicoll, J. A. et al., Nature Med. 9: 448-452
(2003)).
[0019] Recently, a publication has reported 18 cases of
meningoencephalitis in patients immunized by the AN1792, a vaccine
composed of aggregated A.beta.1-42 and QS-21 as adjuvant (Orgogozo
J.-M. et al., Neurology 61: 46-54 (2003)). T-cell activation is
reported as a potential mechanism responsible for the disease,
while there was no clear relation between disease and
anti-A.beta.1-42 titers in the serum.
[0020] It is well established that the administration of purified
proteins alone is usually not sufficient to elicit a strong immune
response; isolated antigen generally must be given together with
helper substances called adjuvants. Within these adjuvants, the
administered antigen is protected against rapid degradation, and
the adjuvant provides an extended release of a low level of
antigen. In the present invention, A.beta. peptides are made
immunogenic through binding to a VLP and do not require an
adjuvant.
[0021] One way to improve the efficiency of vaccination is thus to
increase the degree of repetitiveness of the antigen applied.
Unlike isolated proteins, viruses induce prompt and efficient
immune responses in the absence of any adjuvants both with and
without T-cell help (Bachmann and Zinkernagel, Ann. Rev. Immunol:
15: 235-270 (1991)). Although viruses often consist of few
proteins, they are able to trigger much stronger immune responses
than their isolated components. For B-cell responses, it is known
that one crucial factor for the immunogenicily of viruses is the
repetitiveness and order of surface epitopes. Many viruses exhibit
a quasi-crystalline surface that displays a regular array of
epitopes which efficiently crosslinks epitope-specific
immunoglobulins on B cells (Bachmann and Zinkernagel, Immunol.
Today 17: 553-558 (1996)). This crosslinking of surface
immunoglobulins on B cells is a strong activation signal that
directly induces cell-cycle progression and the production of IgM
antibodies. Further, such triggered B cells are able to activate T
helper cells, which in turn induce a switch from IgM to IgG
antibody production in B cells and the generation of long-lived B
cell memory--the goal of any vaccination (Bachmann and Zinkernagel,
Ann. Rev. Immunol. 15: 235-270 (1997)). Viral structure is even
linked to the generation of anti-antibodies in autoimmune disease
and as a part of the natural response to pathogens (see Fehr, T.,
et al., J Exp. Med. 185: 1785-1792 (1997)). Thus, antibodies
presented by a highly organized viral surface are able to induce
strong anti-antibody responses.
[0022] As indicated, however, the immune system usually fails to
produce antibodies against self-derived structures. For soluble
antigens present at low concentrations, this is due to tolerance at
the Th cell level. Under these conditions, coupling the
self-antigen to a carrier that can deliver T help may break
tolerance. For soluble proteins present at high concentrations or
membrane proteins at low concentration, B and Th cells may be
tolerant. However, B cell tolerance may be reversible (anergy) and
can be broken by administration of the antigen in a highly
organized fashion coupled to a foreign carrier (Bachmann and
Zinkernagel, Ann. Rev. Immunol 15: 235-270 (1997)). As shown in
pending U.S. application Ser. No. 10/050,902 filed on Jan. 18,
2002, strong immune responses could be induced with compositions
comprising A.beta. peptides (A.beta.1-15, A.beta.1-27 and
A.beta.33-42, which is a self-antigen in mice) bound-to a VLP. In
particular, the abovementioned human A.beta. peptides bound to the
VLP of RNA phage Q.beta. induced high A.beta. specific titers in
human APP transgenic mice (described in Example) demonstrating that
tolerance to the self-antigen A.beta. could be overcome by
immunizing with A.beta. peptides bound to a VLP.
[0023] There is thus a need for highly immunogenic safe
compositions and vaccines, respectively, to treat Alzheimer
diseases, in particular, using immunogens devoid of T-cell epitopes
and adjuvants, respectively, which might elicit side-effects, and
still being capable of inducing high antibody titers, which
antibodies, furthermore, being capable of binding to amyloid
plaques.
BRIEF SUMMARY OF THE INVENTION
[0024] We have now found that A.beta.1-6 peptide, which is bound to
a core particle having a structure with an inherent repetitive
organization, and hereby in particular to virus-like-particles
(VLPs) and subunits of VLPs, respectively, leading to highly
ordered and repetitive conjugates represent a potent immunogen for
the induction of antibodies specific for A.beta.1-6. Therefore, the
present invention provides a prophylactic and therapeutic mean for
the treatment of Alzheimer's disease, which is based on an ordered
and repetitive A.beta.1-6 -core particle array, and in particular
on a VLP-A.beta.1-6 peptide conjugate and -array, respectively.
This prophylactic and therapeutic is able to induce high titers of
anti-A.beta.1-6 peptide antibodies. Which are cross-reactive to
soluble A.beta. and are capable of binding to human amyloid plaques
of a human APP transgenic mouse model and to AD amyloid plaques.
Furthermore, the elicited antibodies do not bind to APP on brain
sections.
[0025] Moreover, the present invention provides for new
compositions, vaccines and methods of treatment of AD. The
compositions and vaccines comprising A.beta. 1-6 peptides are
devoid of T-cell epitopes and induce antibodies binding AD plaques
and soluble A.beta.. The A.beta. 1-6 peptides are presented to the
immune system of the patient in a highly repetitive and ordered
fashion through binding of the A.beta. peptides or to a core
particle, preferably to a VLP, and even more preferably to a VLP of
a RNA phage.
[0026] In a preferred embodiment, the antigen or antigenic
determinant is the human amyloid beta peptide A.beta.1-6 (DAEFRH:
SEQ ID NO: 75) being a fragment of A.beta.
(DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 91),
wherein the human amyloid beta peptide A.beta.1-6 is bound to the
core particle and VLP, respectively. The amyloid beta protein is
provided in SEQ ID NO: 92. The amyloid beta precursor protein is
provided in SEQ ID NO: 93.
[0027] The present invention, thus, provides for a composition
comprising: (a) a core particle with at least one first attachment
site; and (b) at least one antigen or antigenic determinant with at
least one second attachment site, wherein said antigen or antigenic
determinant is a A.beta.1-6 peptide, and wherein said second
attachment site being selected from the group consisting of (i) an
attachment site not naturally occurring with said antigen or
antigenic determinant; and (ii) an attachment site naturally
occurring with said antigen or antigenic determinant, wherein said
second attachment site is capable of association to said first
attachment site; and wherein said antigen or antigenic determinant
and said core particle interact through said association to form an
ordered and repetitive antigen array. Preferred embodiments of core
particles suitable for use in the present invention are a virus, a
virus-like particle, a bacteriophage, a virus-like particle of a
RNA-phage, a bacterial pilus or flagella or any other core particle
having an inherent repetitive structure capable of forming an
ordered and repetitive antigen array in accordance with the present
invention.
[0028] The A.beta. fragments of the present invention are soluble
and generally do not form aggregates. Moreover, they are bound, and
preferably covalently bound to a core particle and VLP,
respectively. Therefore, the compositions of the invention do not
bear the risk of inducing toxic effects such as seeding of amyloid
deposition.
[0029] It is an unexpected finding of this invention that a high
titer of antibodies cross-reactive with soluble A.beta. and AD
amyloid plaques could be obtained with a composition comprising the
A.beta.1-6 peptide bound to a a core particle and VLP,
respectively. In particular, VLP have been shown to mediate
induction of antibodies against self antigens, thus breaking
self-tolerance (WO 02/056905, the disclosure of which is herewith
incorporated by reference in its entirety). Furthermore, the small
size of this epitope precludes the presence of T-cell epitopes,
thus providing new compositions that do not induce A.beta. specific
T-cell responses. In addition, the elicited antibodies do not bind
to APP on brain sections. Thus, the present invention provides for
a safe vaccine composition for the prevention and treatment of
AD.
[0030] More specifically, the invention provides a composition
comprising an ordered and repetitive antigen or antigenic
determinant array, and hereby in particular A.beta.1-6 peptide VLP
conjugates. More specifically, the invention provides a composition
comprising a virus-like particle and at least one A.beta.1-6
peptide bound thereto. The invention also provides a process for
producing the conjugates and the ordered and repetitive arrays,
respectively. The compositions of the invention are useful in the
production of vaccines for the treatment of Alzheimer's disease and
as a pharmaceine to prevent or cure Alzheimer's disease and to
efficiently induce immune responses, in particular antibody
responses. Furthermore, the compositions of the invention are
particularly useful to efficiently induce self-specific immune
responses within the indicated context.
[0031] In the present invention, a A.beta.1-6 peptide is bound to a
core particle and VLP, respectively, typically in an oriented
manner, yielding an ordered and repetitive A.beta.1-6 peptide
antigen array. Furthermore, the highly repetitive and organized
structure of the core particles and VLPs, respectively, mediates
the display of the A.beta. peptide in a highly ordered and
repetitive fashion leading to a highly organized and repetitive
antigen array. Furthermore, binding of the A.beta.1-6 peptide to
the core particle and VLP, respectively, provides T helper cell
epitopes, since the core particle and VLP is foreign to the host
immunized with the core particle-A.beta.1-6 peptide array and
VLP-A.beta.1-6 peptide array, respectively. Those arrays differ
from prior art conjugates, in particular, in their highly organized
structure, dimensions, and in the repetitiveness of the antigen on
the surface of the array.
[0032] In one aspect of the invention, the A.beta.1-6 peptide is
chemically synthesized, while the core particle and the VLP,
respectively, is expressed and purified from an expression host
suitable for the folding and assembly of the core particle and the
VLP, respectively. The A.beta.1-6 peptide array is then assembled
by binding the A.beta.1-6 peptide to the core particle and the VLP,
respectively.
[0033] In another aspect, the present invention provides for a
composition comprising (a) a virus-like particle, and (b) at least
one antigen or antigenic determinant, wherein said antigen or said
antigenic determinant is an A.beta.1-6 peptide, and wherein said at
least one antigen or antigenic determinant is bound to said
virus-like particle.
[0034] In a further aspect, the present invention provides for a
pharmaceutical composition comprising (a) the inventive
composition, and (b) an acceptable pharmaceutical carrier.
[0035] In still a further aspect, the present invention provides
for a vaccine composition comprising a composition, wherein said
composition comprising (a) a virus-like particle; and (b) at least
one antigen or antigenic determinant, wherein said antigen or said
antigenic determinant is a A.beta.1-6 peptide; and wherein said at
least one antigen or antigenic determinant is bound to said
virus-like particle.
[0036] In another aspect, the present invention provides for a
method of immunization comprising administering the inventive
composition, the inventive pharmaceutical composition or the
inventive vaccine to an animal.
[0037] In still a further aspect, the present invention provides
for a process for producing an inventive composition comprising (a)
providing a virus-like particle; and (b) providing at least one
antigen or antigenic determinant, wherein said antigen or said
antigenic determinant is a A.beta.1-6 peptide; (c) combining said
virus-like particle and said at least one-antigen or antigenic
determinant so that said at least one antigen or antigenic
determinant is bound to said virus-like particle.
[0038] Analogously, the present invention provides a process for
producing a composition of claim 1 comprising: (a) providing a core
particle with at least one first attachment site; (b) providing at
least one antigen or antigenic determinant with at least one second
attachment site, wherein said antigen or antigenic determinant is a
A.beta.1-6 peptide, and wherein said second attachment site being
selected from the group consisting of (i) an attachment site not
naturally occurring with said antigen or antigenic determinant; and
(ii) an attachment site naturally occurring with said antigen or
antigenic determinant; and wherein said second attachment site is
capable of association to said first attachment site; and (c)
combining said core particle and said at least one antigen or
antigenic determinant, wherein said antigen or antigenic
determinant and said core particle interact through said
association to form an ordered and repetitive antigen array.
[0039] In a further aspect, the present invention provides for a
use of a composition of claim 1 for the manufacture of a medicament
for treatment of Alzheimer's disease.
[0040] In a still further aspect, the present invention provides
for a use of a composition of claim 1 for the preparation of a
medicament for the therapeutic or prophylactic treatment of
Alzheimer's disease. Furthermore, in a still further aspect, the
present invention provides for a use of a composition of claim 1,
either in isolation of in combination with other agents, or with
explicit absence of specific substances such as adjuvants, for the
manufacture of a composition, pharmaceutical composition, vaccine,
drug or medicament for therapy or prophylaxis of Alzheimer's
disease, and/or for stimulating the mammalian immune system.
[0041] Therefore, the invention provides, in particular, vaccine
compositions which are suitable for preventing and/or attenuating
Alzheimer's disease or conditions related thereto. The invention
further provides immunization and vaccination methods,
respectively, for preventing and/or attenuating Alzheimer's disease
or conditions related thereto in humans. The inventive compositions
may be used prophylactically or therapeutically.
[0042] In specific embodiments, the invention provides methods for
preventing and/or attenuating Alzheimer's disease or conditions
related thereto which are caused or exacerbated by "self" gene
products, i.e. "self antigens" as used herein. In related
embodiments, the invention provides methods for inducing
immunological responses in animals and individuals, respectively,
which lead to the production of antibodies that prevent and/or
attenuate Alzheimer's disease or conditions related thereto, which
are caused or exacerbated by "self" gene products.
[0043] As would be understood by one of ordinary skill in the art,
when compositions of the invention are administered to an animal or
a human, they may be in a composition which contains salts,
buffers, adjuvants, or other substances which are desirable for
improving the efficacy of the composition. Examples of materials
suitable for use in preparing pharmaceutical compositions are
provided in numerous sources including Remington's Pharmaceutical
Sciences (Osol, A, ed., Mack Publishing Co. (1990)).
[0044] Compositions of the invention are said to be
"pharmacologically acceptable" if their administration can be
tolerated by a recipient individual. Further, the compositions of
the invention will be administered in a "therapeutically effective
amount" (i.e., an amount that produces a desired physiological
effect).
[0045] The compositions of the present invention may be
administered by various methods known in the art, but will normally
be administered by injection, infusion, inhalation, oral
administration, or other suitable physical methods. The
compositions may alternatively be administered intramuscularly,
intravenously, or subcutaneously. Components of compositions for
administration include sterile aqueous (e.g., physiological saline)
or non-aqueous solutions and suspensions. Examples of non-aqueous
solvents are propylene glycol, polyethylene glycol, vegetable oils
such as olive oil, and injectable organic esters such as ethyl
oleate. Carriers or occlusive dressings can be used to increase
skin permeability and enhance antigen absorption.
[0046] Other embodiments of the present invention will be apparent
to one of ordinary skill in light of what is known in the art, the
following drawings and description of the invention, and the
claims.
BRIEF DESCRIPTION OF VIEWS OF THE FIGURES
[0047] FIG. 1 depicts the SDS-PAGE gel, run under reducing
conditions, showing the result of the coupling of the A.beta.1-6
peptide (NH2-DAEFRHGGC-CONH2) (SEQ ID NO: 77) to the VLP of Q.beta.
coat protein.
[0048] FIG. 2 shows the ELISA analysis of the antibodies specific
for A.beta.1-6 in sera of mice intramixed with A.beta.1-6 peptide
coupled to the VLP of Q.beta. coat protein.
[0049] FIG. 3 shows the ELISA analysis of the antibodies specific
for A.beta.1-40 in sera of mice immunized with A.beta.1-6 peptide
coupled to the VLP of Q.beta. coat protein.
[0050] FIGS. 4 A-B show a brain section of an APP23 mouse (A) and
an entorhinal cortex section from an AD patient (B) stained with
sera of mice immunized with A.beta.1-6 peptide coupled to the VLP
of Q.beta. coat protein.
[0051] FIGS. 5 A-E show brain sections of an APP23 mouse stained
with sera of mice immunized with A.beta.1-6 peptide coupled to the
VLP of Q.beta. coat protein, or with a polyclonal rabbit antiserum
specific for the C-terminus of human or mouse APP.
[0052] FIG. 6 shows the result of the immunization of rhesus
monkeys with human A.beta.1-6 coupled to Q.beta. VLP as measured in
an ELISA assay.
[0053] FIGS. 7 A-B shows the result of the binding to plaques of
sera from monkeys immunized with human A.beta.1-6 coupled to
Q.beta. VLP, as measured by histology on human AD and transgenic
mouse plaques.
[0054] FIG. 8 depicts the SDS-PAGE analysis of the coupling of
murine A.beta.1-6 to AP205 VLP.
[0055] FIG. 9 shows the result of the immunization of mice with
murine A.beta.1-6 coupled to AP205 as measured in an ELISA
assay.
[0056] FIG. 10 shows the analysis by ELISA of the anti-A.beta.40
and anti-A.beta.42 titers in the sera of "Swedish/London"
transgenic mice immunized with Q.beta.hA.beta.1-6 between 9.5 and
19 months of age.
[0057] FIGS. 11 A-B shows the immunohistochemical staining of brain
sections of "Swedish/London" transgenic mice immunized with
Q.beta.hA.beta.1-6 or PBS.
[0058] FIGS. 12 A-H shows the quantification of plaque deposition
in "Swedish/London" transgenic mice, immunized with
Q.beta.hA.beta.1-6, Q.beta. or PBS between 9.5 and 19 months of
age.
[0059] FIGS. 13 A-H shows, the quantification of plaque deposition
in "Swedish/London" transgenic mice immunized with
Q.beta.hA.beta.1-6 or PBS between 13.5 and 19 months of age.
[0060] FIG. 14 shows the analysis by ELISA of the anti-A.beta.40
and anti-A.beta.42 titers in the sera of "Swedish" transgenic mice
immunized with Q.beta.hA.beta.1-6.
[0061] FIGS. 15 A-B shows the immunohistochemical staining of brain
sections from "Swedish" transgenic mice immunized with
Q.beta.hA.beta.1-6 or PBS.
[0062] FIGS. 16 A-B shows the quantification of plaque deposition
in "Swedish" transgenic mice immunized with Q.beta.hA.beta.1-6 or
PBS.
DETAILED DESCRIPTION OF THE INVENTION
[0063] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are hereinafter
described.
1. Definitions:
[0064] A.beta.1-6 peptide: An A.beta.1-6 peptide as used herein
refers to peptides having a sequence corresponding to the human
A.beta.1-6 sequence, or homologous to the human A.beta.1-6
sequence. Sequences homologuous to the human A.beta.1-6 sequence
include, but are hot limited to the A.beta.1-6 sequences of other
species and hereby including, but not limited to, the sequence of
primate, rabbit, guinea pig, Xenopus Laevis, frog, mouse and rat
A.beta.1-6. The A.beta.1-6 sequences from Xenopus Laevis or frog,
although differing from human A.beta.1-6 at two positions, have
conservative mutations (Ala-Ser, Phe-Tyr), and are still considered
to be homologuous to A.beta.1-6 in accordance with this definition.
In accordance with the present invention, however, the A.beta.1-6
peptide is typically modified, such that a second attachment site
is attached thereto. Preferably, the second attachment site is
modified with a linker or an amino acid linker comprising a second
attachment site for binding to a core particle and VLP,
respectively. While referring herein to A.beta.1-6 peptides, a
modified A.beta.1-6 peptide, as indicated above, i.e. A.beta.1-6
peptides with a second attachment site attached thereto, shall be
encompassed. Typically, however, the modifications are explicitly
indicated in the specification. Further preferred embodiments of an
A.beta.1-6 peptide being an antigen or antigenic determinant in
accordance with the present invention become apparent as this
specification proceeds.
[0065] Adjuvant: The term "adjuvant" as used herein refers to
non-specific stimulators of the immune response or substances that
allow generation of a depot in the host which when combined with
the vaccine and pharmaceutical composition, respectively, of the
present invention may provide for an even more enhanced immune
response. A variety of adjuvants can be used. Examples include
complete and incomplete Freund's adjuvant, aluminum hydroxide and
modified muramyldipeptide. Further adjuvants are mineral gels such
as aluminum hydroxide, surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol and
potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are
also well known in the art. Further adjuvants that can be
administered with the compositions of the invention include, but
are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax
100a, QS-21, QS-18, CRL1005, Aluminum salts (Alum), MF-59, OM-174,
OM-197, OM-294, and Virosomal adjuvant technology. The adjuvants
can also comprise a mixture of these substances.
[0066] Immunologically active saponin fractions having adjuvant
activity derived from the bark of the South American tree Quillaja
Saponaria Molina are known in the art. For example QS21, also known
as QA21, is an Hplc purified fraction from the Quillaja Saponaria
Molina tree and it's method of its production is disclosed (as
QA21) in U.S. Pat. No. 5,057,540. Quillaja saponin has also been
disclosed as an adjuvant by Scott et al, Int. Archs. Allergy Appl.
Immun., 1985, 77, 409. Monosphoryl lipid A and derivatives thereof
are known in the art. A preferred derivative is 3 de-o-acylated
monophosphoryl lipid A, and is known from British Patent No.
2220211. Further preferred adjuvants are described in WO00/00462,
the disclosure of which is herein incorporated by reference.
[0067] However, an advantageous feature of the present invention is
the high immunogenicity of the inventive compositions. As already
outlined herein or will become apparent as this specification
proceeds, vaccines and pharmaceutical compositions devoid of
adjuvants are provided, in further alternative or preferred
embodiments, leading to vaccines and pharmaceutical compositions
for treating AD being devoid of adjuvants and, thus, having a
superior safety profile since adjuvants may cause side-effects. The
term "devoid" as used herein in the context of vaccines and
pharmaceutical compositions for treating AD refers to vaccines and
pharmaceutical compositions that are used without adjuvants.
[0068] Amino acid linker: An "amino acid linker", or also just
termed "linker" within this specification, as used herein, either
associates the antigen or antigenic determinant with the second
attachment site, or more preferably, already comprises or contains
the second attachment site, typically--but not necessarily--as one
amino acid residue, preferably as a cysteine residue. The term
"amino acid linker" as used herein, however, does not intend to
imply that such an amino acid linker consists exclusively of amino
acid residues, even if an amino acid linker consisting of amino
acid residues is a preferred embodiment of the present invention.
The amino acid residues of the amino acid linker are, preferably,
composed of naturally securing amino acids or unnatural amino acids
known in the art, all-L or all-D or mixtures thereof. However, an
amino acid linker comprising a molecule with a sulfhydryl group or
cysteine residue is also encompassed within the invention. Such a
molecule comprise preferably a C1-C6 alkyl, cycloalkyl (C5,C6),
aryl or heteroaryl moiety. However, in addition to an amino acid
linker, a linker comprising preferably a C1-C6 alkyl-, cycloalkyl-
(C5,C6), aryl- or heteroaryl- moiety and devoid of any amino
acid(s) shall also be encompassed within the scope of the
invention. Association between the antigen or antigenic determinant
or optionally the second attachment site and the amino acid linker
is preferably by way of at least one covalent bond, more preferably
by way of at least one peptide bond.
[0069] Animal: As used herein, the term "animal" is meant to
include, for example, humans, sheep, elks, deer, mule deer, minks,
mammals, monkeys, horses, cattle, pigs, goats, dogs, cats, rats,
mice, birds, chicken, reptiles, fish, insects and arachnids.
[0070] Antibody: As used herein, the term "antibody" refers to
molecules which are capable of binding an epitope or antigenic
determinant. The term is meant to include whole antibodies and
antigen-binding fragments thereof, including single-chain
antibodies. Most preferably the antibodies are human antigen
binding antibody fragments and include, but are not limited to,
Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain
antibodies, disulfide-linked Fvs (sdFv) and fragments comprising
either a V.sub.L or V.sub.H domain. The antibodies can be from any
animal origin including birds and mammals. Preferably, the
antibodies are human, murine, rabbit, goat, guinea pig, camel,
horse or chicken. As used herein, "human" antibodies include
antibodies having the amino acid sequence of a human immunoglobulin
and include antibodies isolated from human immunoglobulin libraries
or from animals transgenic for one or more human immunoglobulins
and that do not express endogenous immunoglobulins, as described,
for example, in U.S. Pat. No. 5,939,598 by Kucherlapati et al.
[0071] Antigen: As used herein, the term "antigen" refers to a
molecule capable of being bound by an antibody or a T cell receptor
(TCR) if presented by MHC molecules. The term "antigen", as used
herein, also encompasses T-cell epitopes. An antigen is
additionally capable of being recognized by the immune system
and/or being capable of inducing a humoral immune response and/or
cellular immune response leading to the activation of B- and/or
T-lymphocytes. This may, however, require that, at least in certain
cases, the antigen contains or is linked to a Th cell epitope and
is given in adjuvant. An antigen can have one or more epitopes (B-
and T-epitopes). The specific reaction referred to above is meant
to indicate that the antigen will preferably react, typically in a
highly selective manner, with its corresponding antibody or TCR and
not with the multitude of other antibodies or TCRs which may be
evoked by other antigens. Antigens as used herein may also be
mixtures of several individual antigens.
[0072] Antigenic determinant: As used herein, the term "antigenic
determinant" is meant to refer to that portion of an antigen that
is specifically recognized by either B- or T-lymphocytes.
B-lymphocytes responding to antigenic determinants produce
antibodies, whereas T-lymphocytes respond to antigenic determinants
by proliferation and establishment of effector functions critical
for the mediation of cellular and/or humoral immunity.
[0073] Association: As used herein, the term "association" as it
applies to the first and second attachment sites, refers to the
binding of the first and second attachment sites that is preferably
by way of at least one non-peptide bond. The nature of the
association may be covalent, ionic, hydrophobic, polar or any
combination thereof, preferably the nature of the association is
covalent.
[0074] Attachment Site, First: As used herein, the phrase "first
attachment site" refers to an element of non-natural or natural
origin, to which the second attachment site located on the antigen
or antigenic determinant may associate. The first attachment site
may be a protein, a polypeptide, an amino acid, a peptide, a sugar,
a polynucleotide, a natural or synthetic polymer, a secondary
metabolite or compound (biotin, fluorescein, retinol, digoxigenin,
metal ions, phenylmethylsulfonylfluoride), or a combination
thereof, or a chemically reactive group thereof. The first
attachment site is located, typically and preferably on the
surface, of the core particle such as, preferably the virus-like
particle. Multiple first attachment sites are present on the
surface of the core and virus-like particle, respectively,
typically in a repetitive configuration.
[0075] Attachment Site, Second: As used herein, the phrase "second
attachment site" refers to an element associated with the antigen
or antigenic determinant to which the first attachment site located
on the surface of the core particle and virus-like particle,
respectively, may associate. The second attachment site of the
antigen or antigenic determinant may be a protein, a polypeptide, a
peptide, a sugar, a polynucleotide, a natural or synthetic polymer,
a secondary metabolite or compound (biotin, fluorescein, retinol,
digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a
combination thereof, or a chemically reactive group thereof. At
least one second attachment site is present on the antigen or
antigenic determinant. The term "antigen or antigenic determinant
with at least one second attachment site" refers, therefore, to an
antigen or antigenic construct comprising at least the antigen or
antigenic determinant and the second attachment site. However, in
particular for a second attachment site, which is of non-natural
origin, i.e. not naturally occurring within the antigen or
antigenic determinant, these antigen or antigenic constructs
comprise an "amino acid linker".
[0076] Bound: As used herein, the term "bound" refers to binding or
attachment that may be covalent, e.g., by chemically coupling, or
non-covalent, e.g., ionic interactions, hydrophobic interactions,
hydrogen bonds, etc. Covalent bonds can be, for example, ester,
ether, phosphoester, amide, peptide, imide, carbon-sulfur bonds,
carbon-phosphorus bonds, and the like. The term "bound" is broader
than and includes terms such as "coupled," "fused" and
"attached".
[0077] Coat protein(s): As used herein, the term "coat protein(s)"
refers to the protein(s) of a bacteriophage or a RNA-phage capable
of being incorporated within the capsid assembly of the
bacteriophage or the RNA-phage. However, when referring to the
specific gene product of the coat protein gene of RNA-phages the
term "CP" is used. For example, the specific gene product of the
coat protein gene of RNA-phage Q.beta. is referred to as "Q.beta.
CP", whereas the "coat proteins" of bacteriophage Q.beta. comprise
the "Q.beta. CP" as well as the A1 protein. The capsid of
Bacteriophage Q.beta. is composed mainly of the Q.beta. CP, with a
minor content of the A1 protein. Likewise, the VLP Q.beta. coat
protein contains mainly Q.beta. CP, with a minor content of A1
protein.
[0078] Core particle: As used herein, the term "core particle"
refers to a rigid structure with an inherent repetitive
organization. A core particle as used herein may be the product of
a synthetic process or the product of a biological process.
[0079] Coupled: The term "coupled", as used herein, refers to
attachment by covalent bonds or by strong non-covalent
interactions, typically and preferably to attachment by covalent
bonds. Any method normally used by those skilled in the art for the
coupling of biologically active materials can be used in the
present invention.
[0080] Effective Amount: As used herein, the term "effective
amount" refers to an amount necessary or sufficient to realize a
desired biologic effect. An effective amount of the composition
would be the amount that achieves this selected result, and such an
amount could be determined as a matter of routine by a person
skilled in the art. For example, an effective amount for treating
an immune system deficiency could be that amount necessary to cause
activation, of the immune system, resulting in the development of
an antigen specific immune response upon exposure to antigen. The
term is also synonymous with "sufficient amount."
[0081] The effective amount for any particular application can vary
depending on such factors as the disease or condition being
treated, the particular composition being administered, the size of
the subject, and/or the severity of the disease or condition. One
of ordinary skill in the art can empirically determine the
effective amount of a particular composition of the present
invention without necessitating undue experimentation.
[0082] Epitope: As used herein, the term "epitope" refers to
continuous or discontinuous portions of a polypeptide having
antigenic or immunogenic activity in an animal, preferably a
mammal, and roost preferably in a human. An epitope, is recognized
by an antibody or a T cell through its T cell receptor in the
context of an MHC molecule. An "immunogenic epitope," as used
herein, is defined as a portion of a polypeptide that elicits an
antibody response or induces a T-cell response in an animal, as
determined by any method known in the art. (See, for example,
Geysen et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1983)).
The term "antigenic epitope," as used herein, is defined as a
portion of a protein to which an antibody can immunospecifically
bind its antigen as determined by any method well known in the art.
Immunospecific binding excludes non-specific binding but does not
necessarily exclude cross-reactivity with other antigens. Antigenic
epitopes need not necessarily be immunogenic. Antigenic epitopes
can also be T-cell epitopes, in which case they Can be bound
immunospecifically by a T-cell receptor within the context of an
MHC molecule.
[0083] An epitope can comprise 3 amino acids in a spatial
conformation which, is unique to the epitope. Generally, an epitope
consists of at least about 5 such amino acids, and more usually,
consists of at least about 8-10 such amino acids. If the epitope is
an organic molecule, it may be as small as Nitrophenyl.
[0084] Fusion: As used herein, the term "fusion" refers to the
combination of amino acid sequences of different origin in one
polypeptide chain by in-frame combination of their coding
nucleotide sequences. The term "fusion" explicitly encompasses
internal fusions, i.e., insertion of sequences of different origin
within a polypeptide chain, in addition to fusion to one of its
termini.
[0085] Immune response: As used herein, the term "immune response"
refers to a humoral immune response and/or cellular immune response
leading to the activation or proliferation of B- and/or
T-lymphocytes and/or and antigen presenting cells. In some
instances, however, the immune responses may be of low intensity
and become detectable only when using at least one substance in
accordance with the invention. "Immunogenic" refers to an agent
used to stimulate the immune system of a living organism, so that
one or more functions of the immune system are increased and
directed towards the immunogenic agent. An "immunogenic
polypeptide" is a polypeptide that elicits a cellular and/or
humoral immune response, whether alone or linked to a carrier in
the presence or absence of an adjuvant. Preferably, antigen
presenting cell may be activated.
[0086] A substance which "enhances" an immune response refers to a
substance in which an immune response is observed that is greater
or intensified or deviated in any way with the addition of the
substance when compared to the same immune response measured
without the addition of the substance. For example, the lytic
activity of cytotoxic T cells can be measured, e.g. using a
.sup.51Cr release assay, in samples obtained with and without the
use of the substance during immunization. The amount of the
substance at which the CTL lytic activity is enhanced as compared
to the CTL lytic activity without the substance is said to be an
amount sufficient to enhance the immune response of the animal to
the antigen. In a preferred embodiment, the immune response in
enhanced by a factor of at least about 2, more preferably by a
factor of about 3 or more. The amount or type of cytokines secreted
may also be altered. Alternatively, the amount of antibodies
induced or their subclasses may be altered.
[0087] Immunization: As used herein, the terms "immunize" or
"immunization" or related terms refer to conferring the ability to
mount a substantial immune response (comprising antibodies and/or
cellular immunity such as effector CTL) against a target antigen or
epitope. These terms do not require that complete immunity be
created, but rather that an immune response be produced which is
substantially greater than baseline. For example, a mammal may be
considered to be immunised against a target antigen if the cellular
and/or humoral immune response to the target antigen occurs
following the application of methods of the invention.
[0088] Natural origin: As used herein, the term "natural origin"
means that the whole or parts thereof are not synthetic and exist
or are produced in nature.
[0089] Non-natural: As used herein, the term generally means not
from nature, more specifically, the term means from the hand of
man.
[0090] Non-natural origin: As used herein, the term "non-natural
origin" generally means synthetic or not from nature; more
specifically, the term means from the hand of man.
[0091] Ordered and repetitive antigen or antigenic determinant
array: As used herein, the term "ordered and repetitive antigen or
antigenic determinant. array" generally refers to a repeating
pattern of antigen or antigenic determinant, characterized by a
typically and preferably uniform spacial arrangement of the
antigens or antigenic determinants with respect to the core
particle and virus-like particle, respectively. In one embodiment
of the invention, the repeating pattern may be a geometric pattern.
Typical and preferred examples of suitable ordered and repetitive
antigen of antigenic determinant arrays are those which possess
strictly repetitive paracrystalline orders of antigens or antigenic
determinants, preferably with spacings of 0.5 to 30 nanometers,
more preferably 5 to 15 nanometers.
[0092] Pili: As used herein, the term "pili" (singular being
"pilus") refers to extracellular structures of bacterial cells
composed of protein monomers (e.g., pilin monomers) which are
organized into ordered and repetitive patterns. Further, pili are
structures which are involved in processes such as the attachment
of bacterial cells to host cell surface receptors, inter-cellular
genetic exchanges, and cell-cell recognition. Examples of pili
include Type-1 pili, P-pili, F1C pili, S-pili, and 987P-pili.
Additional examples of pili are set out below.
[0093] Pilus-like structure: As used herein, the phrase "pilus-like
structure" refers to structures having characteristics similar to
that of pili and composed of protein monomers. One example of a
"pilus-like structure" is a structure formed by a bacterial cell
which expresses modified pilin proteins that do not form ordered
and repetitive arrays that are identical to those of natural
pili.
[0094] Polypeptide: As used herein, the term "polypeptide" refers
to a molecule composed of monomers (amino acids) linearly linked by
amide bonds (also known as peptide bonds). It indicates a molecular
chain of amino acids and does not refer to a specific length of the
product. Thus, peptides, dipeptides, tripepides, oligopeptides and
proteins are included within the definition of polypeptide. This
term is also intended to refer to post-expression modifications of
the polypeptide, for example, glycosolations, activations,
phosphorylations, and the like. A recombinant or derived
polypeptide is not necessarily translated from a designated nucleic
acid sequence. It may also be generated in any manner, including
chemical synthesis.
[0095] Residue: As used herein, the term "residue" is meant to mean
a specific amino acid in a polypeptide backbone or side chain.
[0096] Self antigen: As used herein, the tern "self antigen" refers
to proteins encoded by the host's DMA and products generated by
proteins or RNA encoded by the host's DMA are defined as self. In
addition, proteins that result from a combination of two or several
self-molecules or that represent a fraction of a self-molecule and
proteins that have a high homology two self-molecules as defined
above (>95%, preferably >97%, more preferably >99%) may
also be considered self.
[0097] Treatment: As used herein, the terms "treatment", "treat",
"treated" or "treating" refer to prophylaxis, and/or therapy. When
used with respect to an infectious disease, for example, the term
refers to a prophylactic treatment which increases the resistance
of a subject to infection with a pathogen or, in other words,
decreases the likelihood that the subject will become infected with
the pathogen or will show signs of illness attributable to the
infection, as well as a treatment after the subject has become
infected in order to fight the infection, e.g., reduce or eliminate
the infection or prevent it from becoming worse.
[0098] Vaccine: As used herein, the term "vaccine" refers to a
formulation which contains the composition of the present invention
and which is in a form that is capable of being administered to an
animal. Typically, the vaccine comprises a conventional saline or
buffered aqueous solution medium in which the composition of the
present invention is suspended or dissolved. In this form, the
composition of the present invention can be used conveniently to
prevent, ameliorate, or otherwise treat a condition. Upon
introduction into a host, the vaccine is able to provoke an immune
response including, but not limited to, the production of
antibodies and/or cytokines and/or the activation of cytotoxic T
cells, antigen presenting cells, helper T cells, dendritic cells
and/or other cellular responses.
[0099] Optionally, the vaccine of the present invention
additionally includes an adjuvant which can be present in either a
minor or major proportion relative to the compound of the present
invention.
[0100] Virus-like particle (VLP): As used herein, the term
"virus-like particle" refers to a structure resembling a virus
particle. Moreover, a virus-like particle in accordance with the
invention is non replicative and noninfectious since it lacks all
or part of the viral genome. In particular the replicative and
infectious components of the viral genome. A virus-like particle in
accordance with the invention may contain nucleic acid distinct
front their genome. A typical and preferred embodiment of a
virus-like particle in accordance with the present invention is a
viral capsid such as the viral capsid of the corresponding virus,
bacteriophage, or RNA-phage. The terms "viral capsid" or "capsid",
as interchangeably used herein, refer to a macromolecular assembly
composed of viral protein subunits. Typically and preferably, the
viral protein subunits assemble into a viral capsid and capsid,
respectively, having a structure with an inherent repetitive
organization, wherein said structure is, typically, spherical or
tubular. For example, the capsids of RNA-phages or HBcAg's have a
spherical form of icosahedral symmetry. The term "capsid-like
structure" as used herein, refers to a macromolecular assembly
composed of viral protein subunits ressembling the capsid
morphology in the above defined sense but deviating from the
typical symmetrical assembly while maintaining a sufficient degree
of order and repetitiveness.
[0101] Virus-like particle of a bacteriophage: As used herein, the
term "virus-like particle of a bacteriophage" refers to a
virus-like particle resembling the structure of a bacteriophage,
being non replicative and noninfectious, and lacking at least the
gene or genes encoding for the replication machinery of the
bacteriophage, and typically also lacking the gene or genes
encoding the protein or proteins responsible for viral attachment
to or entry into the host. This definition should, however, also
encompass virus-like particles of bacteriophages, in which the
aforementioned gene or genes are still present but inactive, and,
therefore, also leading to non-replicative and noninfectious
virus-like particles of a bacteriophage.
[0102] VLP of RNA phage coat protein: The capsid structure formed
from the self-assembly of 180 subunits of RNA phage coat protein
and optionally containing host RNA is referred to as a "VLP of RNA
phage coat protein". A specific example is the VLP of Q.beta. coat
protein. In this particular case, the VLP of Q.beta. coat protein
may either be assembled exclusively from Q.beta. CP subunits
(generated by expression of a Q.beta. CP gene containing, for
example, a TAA stop codon precluding any expression of the longer
A1 protein through suppression, see Kozlovska, T. M., et al.,
Intervirology 39: 9-15 (1996)), or additionally contain A1 protein
subunits in the capsid assembly.
[0103] Virus particle: The term "virus particle" as used herein
refers to the morphological form of a virus. In some virus types it
comprises a genome surrounded by a protein capsid; others have
additional structures (e.g., envelopes, tails, etc.).
[0104] One, a, or an: When the terms "one," "a," or "an" are used
in this disclosure, they mean "at least one" or "one or more,"
unless otherwise indicated.
[0105] As will be clear to those skilled in the art, certain
embodiments of the invention involve the use of recombinant nucleic
acid technologies such as cloning, polymerase chain reaction, the
purification of DNA and RNA, the expression of recombinant proteins
in prokaryotic and eukaryotic cells, etc. Such methodologies are
well known to those skilled in the art and can be conveniently
found in published laboratory methods manuals (e.g., Sambrook, J.
et al., eds., Molecular Cloning, A laboratory Manual 2nd. edition,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989); Ausubel, F. et al., eds., Current Protocols in Molecular
Biology, John H. Wiley & Sons, Inc. (1997)). Fundamental
laboratory techniques for working with tissue culture cell lines
(Cells, J., ed., Cell Biology, Academic Press, 2.sup.nd edition,
(1998)) and antibody-based technologies (Harlow, E. and Lane, D.,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y. (1988); Deutscher, M. P., "Guide to
Protein Purification," Meth. Enzymol. 128, Academic Press San Diego
(1990); Scopes, R. K., Protein Purification Principles and
Practice, 3rd ed., Springer-Verlag, New York (1994)) are also
adequately described in the literature, all of which are
incorporated herein by reference.
[0106] 2. Compositions and Methods for Enhancing an Immune
Response
[0107] The disclosed invention provides compositions and methods
for inducing an immune response against A.beta.1-6 peptide in an
animal, inducing antibodies capable of binding A.beta. amyloid
plaques and soluble A.beta.. Compositions of the invention
comprise, or alternatively consist of (a) a core particle with at
least one first attachment site; and (b) at least one antigen or
antigenic determinant with at least one second attachment site,
wherein said antigen or antigenic determinant is an A.beta.1-6
peptide, and wherein said second attachment site being selected
from the group consisting of (i) an attachment site not naturally
occurring with said antigen or antigenic determinant; and (ii) an
attachment site naturally occurring with said antigen or antigenic
determinant, wherein said second attachment site is capable of
association to said first attachment site; and wherein said antigen
or antigenic determinant and said core particle interact through
said association to form an ordered and repetitive antigen array.
More specifically, compositions of the invention comprise, or
alternatively consist of, a virus-like particle and at least one
antigen or antigenic determinant, wherein the antigen or antigenic
determinant is a A.beta.1-6 peptide, and wherein the at least one
antigen or antigenic determinant is bound to the virus-like
particle so as to form an ordered and repetitive antigen-VLP-array.
Furthermore, the invention conveniently enables the practitioner to
construct such a composition, inter alia, for treatment and/or
prophylactic, prevention of Alzheimer s disease. Virus-like
particles in the context of the present application refer to
structures resembling a virus particle but which are not
pathogenic. In general, virus-like particles lack the viral genome
and, therefore, are noninfectious. Also, virus-like particles can
be produced in large quantities by heterologous expression and can
be easily purified.
[0108] In one embodiment, the core particle comprises, or is
selected from a group consisting of, a virus, a bacterial pilus, a
structure formed from bacterial pilin, a bacteriophage, a
virus-like particle, a virus-like particle of a RNA phage, a viral
capsid particle or a recombinant form thereof. Any virus known in
the art having an ordered and repetitive coat and/or core protein
structure may be selected as a core particle of the invention;
examples of suitable viruses include sindbis and other
alphaviruses, rhabdoviruses: (e.g. vesicular stomatitis virus),
picornaviruses (e.g., human rhino virus, Aichi virus), togaviruses
(e.g., rubella virus), orthomyxoviruses (e.g., Thogoto virus,
Batken virus, fowl plague virus), polyomaviruses (e.g.,
polyomavirus BK, polyomavirus JC, avian polyomavirus BFDV),
parvoviruses, rotaviruses. Norwalk virus, foot and mouth disease
virus, a retrovirus, Hepatitis B virus, Tobacco mosaic virus, Flock
House Virus, and human Papilomavirus, and preferably a RNA phage,
bacteriophage Q.beta., bacteriophage R17, bacteriophage M11,
bacteriophage MX1, bacteriophage NL95, bacteriophage fr,
bacteriophage GA, bacteriophage SP, bacteriophage MS2,
bacteriophage f2, bacteriophage PP7 (for example, see Table 1 in
Bachmann, M. F. and Zinkernagel, R. M., Immunol Today 17: 553-558
(1996)).
[0109] In a further embodiment, the invention utilizes genetic
engineering of a virus to create a fusion between an ordered and
repetitive viral envelope protein and a first attachment site being
comprised by, or alternatively or preferably being a heterologous
protein, peptide, antigenic determinant or a reactive amino acid
residue of choice. Other genetic manipulations known to those in
the art may be included in the construction of the inventive
compositions; for example, it may be desirable to restrict the
replication ability of the recombinant virus through genetic
mutation. Furthermore, the virus used for the present invention, is
replication incompetent due to chemical or physical inactivation
or, as indicated, due to lack of a replication competent genome.
The viral protein selected for fusion to the first attachment site
should have an organized and repetitive structure. Such an
organized and repetitive structure includes paracrystalline
organizations with a spacing of 5-30 nm, preferably 5-15 nm, on the
surface of the virus. The creation of this type of fusion protein
will result in multiple, ordered and repetitive first attachment
sites on the surface of the virus and reflect the normal
organization of the native viral protein. As will be understood by
those in the art, the first attachment site may be or be a part of
any suitable protein, polypeptide, sugar, polynucleotide, peptide
(amino acid), natural or synthetic polymer, a secondary metabolite
or combination thereof that may serve to specifically attach the
antigen or antigenic determinant leading an ordered and repetitive
antigen array.
[0110] In another embodiment of the invention, the core particle is
a recombinant alphavirus, and more specifically, a recombinant
Sinbis virus. Alphaviruses are positive stranded RNA viruses that
replicate their genomic RNA entirely in the cytoplasm of the
infected cell and without a DNA intermediate (Strauss, J. and
Strauss, E., Microbiol Rev. 58: 491-562 (1994)). Several members of
the alphavirus family, Sindbis (Xiong, C. et al., Science 243:
1188-1191 (1989); Schlesinger, S., Trends Biotechnol. 11:18-22
(1993)), Semliki Forest Virus (SFV) (Liljestrom, P. & Garoff,
H., Bio/Technology 9: 1356-1361 (1991)) and others (Davis, N. L. et
al., Virology 171: 189-204 (1989)), have received considerable
attention for use as virus-based expression vectors for a variety
of different proteins (Lundstrom, K., Curr. Opin. Biotechnol. 8:
578-582 (1997); Liljestrom, P., Curr. Opin. Biotechnol. 5: 495-500
(1994)) and as candidates for vaccine development. Recently, a
number of patents have issued directed to the use of alphaviruses
for the expression of heterologous proteins and the development of
vaccines (see U.S. Pat. Nos. 5,766,602; 5,792,462: 5,739,026;
5,789,245 and 5,814,482). The construction of the alpha viral core
particles of the invention may be done by means generally known in
the art of recombinant DNA technology, as described by the
aforementioned articles, which are incorporated herein by
reference.
[0111] A variety of different recombinant host cells can be
utilized to produce a viral-based core particle for antigen or
antigenic determinant attachment. For example, alphaviruses are
known to have a wide host range; Sindbis virus infects cultured
mammalian, reptilian, and amphibian cells, as well as some insect
cells (Clark, H., J. Natl. Cancer Inst. 51: 645 (1973): Leake, C.,
J. Gen. Virol. 35: 335 (1977); Stollar, V. in THE TOGAVIRUSES, R.
W. Schlesinger, Ed., Academic Press, (1980), pp. 583-621). Thus,
numerous recombinant host cells can be used in the practice of the
invention, BHK, COS, Vero, HeLa and CHO cells are particularly
suitable for the production of heterologous proteins because they
have the potential to glycosylate heterologous proteins in a manner
similar to human cells (Watson, E. et al., Glycobiology 4: 227,
(1994)) and can be selected (Zang, M. et al., Bio/Technology 13:
389 (1995)) or genetically engineered (Renner W. et al., Biotech.
Bioeng. 4:476 (1995); Lee K. et al. Biotech. Bioeng. 50: 336
(1996)) to grow in serum-free medium, as well as in suspension.
[0112] Introduction of the polynucleotide vectors into host cells
can be effected by methods described in standard laboratory manuals
(see, e.g., Sambrook J. et al., eds., MOLECULAR CLONING, A
LABORATORY MANUAL, 2nd, edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1989), Chapter 9; Ausubel, F. et
al., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley
& Sons, Inc. (1997), Chapter 16), including methods such as
electroporation, DEAE-dextran mediated transaction, transfection,
microinjection, cationic lipid-mediated transfection, transduction,
scrape loading, ballistic introduction, and infection. Methods for
the introduction of exogenous DNA sequences into host cells are
discussed in Feigner, P. et al., U.S. Pat. No. 5,580,859.
[0113] Packaged RNA sequences can also be used to infect host
cells. These packaged RNA sequences can be introduced to host cells
by adding them to the culture medium. For example, the preparation
of non-infective alphaviral particles is described in a number of
sources, including "Sindbis Expression System", Version C
(Invitrogen Catalog No. K750-1).
[0114] When mammalian cells are used as recombinant host cells for
the production of viral-based core particles, these cells will
generally be grown in tissue culture. Methods for growing cells in
culture are well known in the art (see, e.g., Cells, J., ed., CELL
BIOLOGY, Academic Press, 2.sup.nd edition, (1998); Sambrook, J. et
al., eds., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd, edition,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y,
(1989); Ausubel, F. et al., eds., CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY. John H. Wiley & Sons, Inc. ((997); Freshney, R.,
CULTURE OF ANIMAL CELLS, Alan R, Liss. Inc. (1983)).
[0115] Further examples of RNA viruses suitable for use as core
particle in the present invention include, but are not limited to,
the following: members of the family Reoviridae, including the
genus Orthoreovirus (multiple serotypes of both mammalian and avian
retroviruses), the genus Orbivirus (Bluetongue virus, Eugenangee
virus, Kemerovo virus, African horse sickness virus, and Colorado
Tick Fever virus), the genus Rotavirus (human rotavirus, Nebraska
calf diarrhea virus, murine rotavirus, simian rotavirus, bovine or
ovine rotavirus, avian rotavirus); the family Picomaviridae,
including the genus Enterovirus (poliovirus, Coxsackie virus A and
B, enteric cytopathic human orphan (ECHO) viruses, hepatitis A, C,
D, E and G viruses, Simian enteroviruses, Murine encephalomyelitis
(ME) viruses, Poliovirus muris, Bovine enteroviruses, Porcine
enteroviruses, the genus Cardiovirus (Encephalomyocarditis virus
(EMC), Mengovirus), the genus Rhinovirus (Human rhinoviruses
including at least 113 subtypes; other rhinoviruses), the genus
Apthovirus (Foot and Mouth disease (FMDV); the family Calciviridae,
including Vesicular exanthema of swine virus, San Miguel sea lion
virus, Feline picornavirus and Norwalk virus; the family
Togaviridae, including the genus Alphavirus (Eastern equine
encephalitis virus, Semliki forest virus, Sindbis virus,
Chikungunya virus, O'Nyong-Nyong virus. Ross river virus,
Venezuelan equine encephalitis virus. Western equine encephalitis
virus), the genus Flavirius (Mosquito borne yellow fever virus,
Dengue virus, Japanese encephalitis virus, St Louis encephalitis
virus, Murray Valley encephalitis virus, West Nile virus, Kunjin
virus, Central European tick borne virus, Far Eastern tick borne
virus, Kyasanur forest virus, Louping III virus, Powassan virus,
Omsk hemorrhagic fever virus), the genus Rubivirus (Rubella virus),
the genus Pestivirus (Mucosal disease virus, Hog cholera virus,
Border disease virus); the family Bunyaviridae, including the genus
Bunyvirus (Bunyamwera and related viruses, California encephalitis
group viruses), the genus Phlebovirus (Sandfly fever Sicilian
virus, Rift Valley fever virus), the genus Nairovirus
(Crimean-Congo hemorrhagic fever virus, Nairobi sheep disease
virus), and the genus Uukuvirus (Uukuniemi and related viruses);
the family Orthomyxoviridae, including the genus Influenza virus
(Influenza virus type A, many human subtypes); Swine influenza
virus, and Avian and Equine Influenza viruses; influenza type B
(many human subtypes), and influenza type C (possible separate
genus); the family paramyxoviridae, including the genus
Paramyxovirus (Parainfluenza virus type 1, Sendai virus,
Hemadsorption virus, Parainfluenza viruses types 2 to 5, Newcastle
Disease Virus, Mumps virus), the genus Morbillivirus (Measles
virus, subacute sclerosing panencephalitis virus, distemper virus,
Rinderpest virus), the genus Pneumovirus (respiratory syncytial
virus (RSV), Bovine respiratory syncytial virus and Pneumonia virus
of mice); forest virus, Sindbis virus, Chikangunya virus,
O'Nyong-Nyong virus, Ross river virus, Venezuelan equine
encephalitis virus, Western equine encephalitis virus), the genus
Flavirius (Mosquito borne yellow fever virus, Dengue virus,
Japanese encephalitis virus, St. Louis encephalitis virus, Murray
Valley encephalitis virus, West Nile virus, Kunjin virus, Central
European tick borne virus. Far Eastern tick borne virus, Kyasanur
forest virus, Louping III virus, Powassan virus, Omsk hemorrhagic
fever virus), the genus Rubi virus (Rubella virus), the genus
Pestivirus (Mucosal disease virus, Hog cholera virus, Border
disease virus); the family Bunyaviridae, including the genus
Bunyvirus (Bunyamwera and related viruses, California encephalitis
group viruses), the genus Phlebovirus (Sandfly fever Sicilian
virus, Rift Valley fever virus), the genus Nairovirus
(Crimean-Congo hemorrhagic fever virus, Nairobi sheep disease
virus), and the genus Uukuvirus (Unkuniemi and related viruses);
the family Orthomyxoviridae, including the genus Influenza virus
(Influenza virus type A, many human subtypes); Swine influenza
virus, and Avian and Equine Influenza viruses; influenza type B
(many human subtypes), and influenza type C (possible separate
genus); the family paramyxoviridae, including the genus
Paramyxovirus (Parainfluenza virus type 1, Sendai virus,
Hemadsorption virus, Parainfluenza viruses types 2 -to 5, Newcastle
Disease Virus, Mumps virus), the genus Morbillivirus (Measles
virus, subacute sclerosing panencephalitis virus, distemper virus,
Rinderpest virus), the genus Pneumovirus (respiratory syncytial
virus (RSV), Bovine respiratory syncytial virus and Pneumonia virus
of mice); the family Rhabdoviridae, including the genus
Vesiculovirus (VSV), Chandipura virus, Flanders-Hart Park virus),
the genus Lyssavirus (Rabies virus), fish Rhabdoviruses and,
filoviruses (Marburg virus and Ebola virus); the family
Arenaviridae, including Lymphocytic choriomeningitis virus (LCM),
Tacaribe virus complex, and Lassa virus; the family Coronoaviridae,
including Infectious Bronchitis Virus (IBV), Mouse Hepatitis virus,
Human enteric corona virus, and Feline infectious peritonitis
(Feline coronavirus).
[0116] Illustrative DNA viruses that may be used as core particle
include, but are not limited to: the family Poxviridae, including
the genus Orthopoxvirus (Variola major, Variola minor, Monkey pox
Vaccinia, Cowpox, Buffalopox. Rabbitpox, Ectromelia), the genus
Leporipoxvirus (Myxoma, Fibroma), the genus A vipoxvirus (Fowlpox,
other avian poxvirus), the genus Capripoxvirus (sheeppox, goatpox),
the genus Suipoxvirus (Swinepox), the genus Parapoxvirus
(contagious postular dermatitis virus, pseudocowpox, bovine papular
stomatitis virus); the family Iridoviridae (African swine fever
virus. Frog viruses 2 and 3, Lymphocystis virus of fish); the
family Herpesviridae, including the alpha-Herpesviruses (Herpes
Simplex Types 1 and 2, Varicella-Zoster, Equine abortion virus,
Equine herpes virus 2 and 3, pseudorabies virus, infectious bovine
keratoconjunctivitis virus, infectious bovine rhinotracheitis
virus, feline rhinotracheitis virus, infectious laryngotracheitis
virus) the Beta-herpesviruses (Human cytomegalovirus and
cytomegaloviruses of swine, monkeys and rodents); the
gamma-herpesviruses (Epstein-Barr virus (EBV), Marek's disease
virus, Herpes saimiri, Herpesvirus ateles, Herpesvirus sylvilagus,
guinea pig herpes virus, Lucke tumor virus); the family
Adenoviridae, including the genus Mastadenovirus (Human subgroups
A, B, C, D and E and ungrouped; simian adenoviruses (at least 23
serotypes), infectious canine hepatitis, and adenoviruses of
cattle, pigs, sheep, frogs and many other species, the genus
Aviadenovirus (Avian adenoviruses); and non-cultivatable
adenoviruses; the family Papoviridae, including the genus
Papillomavirus (Human papilloma viruses, bovine papilloma viruses,
Shope rabbit papilloma virus, and various pathogenic papilloma
viruses of other species), the genus Polyomavirus (polyomavirus,
Simian vacuolating agent (SV-40), Rabbit vacuolating agent (RKV), K
virus, BK virus, JC virus, and other primate polyoma viruses such
as Lymphotrophic papilloma virus); the family Parvoviridae
including the genus Adeno-associated viruses, the genus Parvovirus
(Feline panleukopenia virus, bovine parvovirus, canine parvovirus,
Aleutian mink disease virus, etc.). Finally, DNA viruses may
include viruses such as chronic infections neuropathic agents
(CHINA virus).
[0117] In other embodiments, a bacterial pilin, a subportion of a
bacterial pilin, or a fusion protein which contains either a
bacterial pilin or subportion thereof is used to prepare
compositions and vaccine compositions, respectively, of the
invention. Examples of pilin proteins include pilins produced by
Escherichia coli, Haemophilus influenzae, Neisseria meningitidis,
Neisseria gonorrhoeae, Caulobacter crescentus, Pseudomonas
stutzeri, and Pseudomonas aeruginosa. The amino acid sequences of
pilin proteins suitable for use with the present invention include
those set out in GenBank reports AJ000636, AJ132364, AF229646,
AF051814, AF051815, and X00981, the entire disclosures of which are
incorporated herein by reference.
[0118] Bacterial pilin proteins are generally processed to remove
N-terminal leader sequences prior to export of the proteins into
the bacterial periplasm. Further, as one skilled in the art would
recognize, bacterial pilin proteins used to prepare compositions
and vaccine compositions, respectively, of the invention will
generally not have the naturally present leader sequence.
[0119] One specific example of a pilin protein suitable for use in
the present invention is the P-pilin of E. coli (GenBank report
AF237482 (SEQ ID NO:1)). An example of a Type-1 E. coli pilin
suitable for use with the invention is a pilin having the amino
acid sequence set out in GenBank report P04128 (SEQ ID NO:2), which
is encoded by nucleic acid having the nucleotide sequence set out
in GenBank report M27603 (SEQ ID NO:3). The entire disclosures of
these GenBank reports are incorporated herein by reference. Again,
the mature form of the above referenced protein would generally be
used to prepare compositions and vaccine compositions,
respectively, of the invention.
[0120] Bacterial pilins or pilin subportions suitable for use in
the practice of the present invention will generally be able to
associate to form ordered and repetitive antigen arrays.
[0121] Methods for preparing pili and pilus-like structures in
vitro are known in the art. Bullitt et al., Proc. Natl. Acad. Sci.
USA 93: 12890-12895 (1996), for example, describe the in vitro
reconstitution of E. coli P-pili subunits. Furthermore, Eshdat et
al., J. Bacteriol. 148: 308-314 (1981) describe methods suitable
for dissociating Type-1 pili of E. coli and the reconstitution of
pili. In brief, these methods are as follows: pili are dissociated
by incubation at 37.degree. C. in saturated guanidine
hydrochloride. Pilin proteins are then purified by chromatography,
after which pilin dimers are formed by dialysis against 5 mM
tris(hydroxymethyl) aminomethane hydrochloride (pH 8.0). Eshdat et
al. also found that pilin dimers reassemble to form pili upon
dialysis against the 5 mM tris(hydroxymethyl) aminomethane (pH 8.0)
containing 5 mM MgCl.sub.2.
[0122] Further, using, for example, conventional genetic
engineering and protein modification methods, pilin proteins may be
modified to contain a first attachment site to which an antigen or
antigenic determinant is linked through a second attachment site.
Alternatively, antigens or antigenic determinants can be directly
linked through a second attachment site to amino acid residues
which are naturally resident in these proteins. These modified
pilin proteins may then be used in vaccine compositions of the
invention.
[0123] Bacterial pilin proteins used to prepare compositions and
vaccine compositions, respectively, of the invention may be
modified in a manner similar to that described herein for HBcAg.
For example, cysteine and lysine residues may be either deleted or
substituted with other amino acid residues and first attachment
sites may be added to these proteins. Further, pilin proteins may
either be expressed in modified form or may be chemically modified
after expression. Similarly, intact pili may be harvested from
bacteria and then modified chemically.
[0124] In another embodiment, pili or pilus-like structures are
harvested from bacteria (e.g., E. coli) and used to form
compositions and vaccine compositions of the invention. One example
of pili suitable for preparing compositions and vaccine
compositions is the Type-1 pilus of E. coli, which is formed from
pilin monomers having the amino acid sequence set out in SEQ ID
NO:2.
[0125] A number of methods for harvesting bacterial pili are known
in the art. Bullitt and Makowski (Biophys. J. 74: 623-632 (1998)),
for example, describe a pilus purification method for harvesting
P-pili from E. coli. According to this method, pili are sheared
from hyperpiliated E. coli containing a P-pilus plasmid and
purified by cycles of solubilization and MgCl.sub.2 (1.0 M)
precipitation.
[0126] Once harvested, pili or pilus-like structures may be
modified in a variety of ways. For example, a first attachment site
can be added to the pili to which antigens or antigen determinants
may be attached through a second attachment site. In other words,
bacterial pili or pilus-like structures can be harvested and
modified to lead to ordered and repetitive antigen arrays.
[0127] Antigens or antigenic determinants could be linked to
naturally occurring cysteine resides or lysine residues present in
Pill or pilus-like structures. In such instances, the high order
and repetitiveness of a naturally occurring amino acid residue
would guide the coupling of the antigens or antigenic determinants
to the pili or pilus-like structures. For example, the pili or
pilus-like structures could be linked to the second attachment
sites of the antigens or antigenic determinants using a
heterobifunctional cross-linking agent.
[0128] When structures which are naturally synthesized by organisms
(e.g., pili) are used to prepare compositions and vaccine
compositions of the invention, it will often be advantageous to
genetically engineer these organisms so that they produce
structures having desirable characteristics. For example, when
Type-1 pili of E. coli are used, the E. coli from which these pili
are harvested may be modified so as to produce structures with
specific characteristics. Examples of possible modification's of
pilin proteins include the insertion of one or more lysine
residues, the deletion or substitution of one or more of the
naturally resident lysine residues, and the deletion or
substitution of one or more naturally resident cysteine residues
(e.g., the cysteine residues at positions 44 and 84 in SBQ ID
NO:2).
[0129] Further, additional modifications can be made to pilin genes
which result in the expression products containing a first
attachment site other than a lysine residue (e.g., a FOS or JUN
domain). Of course, suitable first attachment sites will generally
be limited to those which do not prevent pilin proteins from
forming pili or pilus-like structures suitable for use in vaccine
compositions of the invention.
[0130] Pilin genes which naturally reside in bacterial cells can be
modified in vivo (e.g., by homologous recombination) or pilin genes
with particular characteristics can be inserted into these cells.
For examples, pilin genes could be introduced into bacterial cells
as a component of either a replicable cloning vector or a vector
which inserts into the bacterial chromosome. The inserted pilin
genes may also be linked to expression regulatory control sequences
(e.g., a lac operator).
[0131] In most instances, the pili or pilus-like structures used in
compositions and vaccine compositions, respectively, of the
invention will be composed of single type of a pilin subunit. Pili
or pilus-like structures composed of identical subunits will
generally be used because they are expected to form structures
which present highly ordered and repetitive antigen arrays.
[0132] However, the compositions of the invention also include
compositions and vaccines comprising pili, or pilus-like structures
formed from heterogenous pilin subunits. The pilin subunits which
form these pili or pilus-like structures can be expressed from
genes naturally resident in the bacterial cell or may be introduced
into the cells. When a naturally resident pilin gene and an
introduced gene are both expressed in a cell which forms pili or
pilus-like structures, the result will generally be structures
formed from a mixture of these pilin proteins. Further, when two or
more pilin genes are expressed in a bacterial cell, the relative
expression of each pilin gene will typically be the factor which
determines the ratio of the different pilin subunits in the pili or
pilus-like structures.
[0133] When pili or pilus-like structures having a particular
composition of mixed pilin subunits is desired, the expression of
at least one of the pilin genes can be regulated by a heterologous,
inducible promoter. Such promoters, as well as other genetic
elements, can be used to regulate the relative amounts of different
pilin subunits produced in the bacterial cell and, hence, the
composition of the pili or pilus-like structures.
[0134] In additional, the antigen or antigenic determinant can be
linked to bacterial pili or pilus-like structures by a bond which
is not a peptide bond, bacterial cells which produce pili or
pilus-like structures used in the compositions of the invention can
be genetically engineered to generate pilin proteins which are
fused to an antigen or antigenic determinant. Such fusion proteins
which form pili or pilus-like structures are suitable for use in
vaccine compositions of the invention.
[0135] In a preferred embodiment, the core particle is a virus-like
particle, wherein the virus-like particle is a recombinant
virus-like particle. The skilled artisan can produce VLPs using
recombinant DNA technology and virus coding sequences which are
readily available to the public. For example, the coding sequence
of a virus envelope or core protein can be engineered for
expression in a baculovirus expression vector using a commercially
available baculovirus vector, under the regulatory control of a
virus promoter, with appropriate modifications of the sequence to
allow functional linkage of the coding sequence to the regulatory
sequence. The coding sequence of a virus envelope or core protein
can also be engineered for expression in a bacterial expression
vector, for example.
[0136] Examples of VLPs include, but are not limited to, the capsid
proteins of Hepatitis B virus (Ulrich, et al., Virus Res. 50:
141-182 (1998)), measles virus (Warnes, et al., Gene 160: 173-178
(1995)), Sindbis virus, rotavirus (U.S. Pat. No. 5,071,651 and U.S.
Pat. No. 5,374,426), foot-and-mouth-disease virus (Twomey, et al.,
Vaccine 13: 1603-1610, (1995)), Norwalk virus (Jiang, X., et al.,
Science 250: 1580-1583 (1990); Matsui, S. M., et al., J. Clin.
Invest. 87: 1456-1461 (1991)), the retroviral GAG protein (WO
96/30523), the retotransposon Ty protein p1, the surface protein of
Hepatitis B virus (WO 92/11291), human papilloma virus (WO
98/15631), RNA phages, Ty, fr-phage, GA-phage, AP205-phage and
Q.beta.-phage.
[0137] As will be readily apparent to those skilled in the art, the
VLP of the invention is not limited to any specific form. The
particle can be synthesized chemically or through a biological
process, which can be natural or non-natural. By way of example,
this type of embodiment includes a virus-like particle or a
recombinant form thereof.
[0138] In a more specific embodiment, the VLP can comprise, or
alternatively essentially consist of, or alternatively consist of
recombinant polypeptides, or fragments thereof being selected from
recombinant polypeptides of Rotavirus, recombinant polypeptides of
Norwalk virus, recombinant polypeptides of Alphavirus, recombinant
polypeptides of Foot and Mouth Disease virus, recombinant
polypeptides of measles virus, recombinant polypeptides of Sindbis
virus, recombinant polypeptides of Polyoma virus, recombinant
polypeptides of Retrovirus, recombinant polypeptides of Hepatitis B
virus (e.g., a HBcAg), recombinant polypeptides of Tobacco mosaic
virus, recombinant polypeptides of Flock House Virus, recombinant
polypeptides of human Papillomavirus, recombinant polypeptides of
bacteriophages, recombinant polypeptides of RNA phages, recombinant
polypeptides of Ty, recombinant polypeptides of fr-phage,
recombinant polypeptides of GA-phage and recombinant polypeptides
of Q.beta.-phage. The virus-like particle can further comprise, or
alternatively essentially consist of, or alternatively consist of,
one or more fragments of such polypeptides, as well as variants of
such polypeptides. Variants of polypeptides can share, for example,
at least 80%, 85%, 90%, 95%, 97%, or 99% identity at the amino acid
level with their wild-type counterparts.
[0139] In a preferred embodiment, the virus-like particle
comprises, consists essentially of, or alternatively consists of
recombinant proteins, or fragments thereof, of a RNA-phage.
Preferably, the RNA-phage is selected from the group consisting of
a) bacteriophage Q.beta.; b) bacteriophage R17; c) bacteriophage
fr; d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2;
g) bacteriophage M11; h) bacteriophage MX1; i) bacteriophage NL95;
k) bacteriophage f2; l) bacteriophage PP7, and m) bacteriophage
AP205.
[0140] In another preferred embodiment of the present invention,
the virus-like particle comprises, or alternatively consists
essentially of, or alternatively consists of recombinant proteins,
or fragments thereof of the RNA-bacteriophage Q.beta. or of the
RNA-bacteriophage fr, or of the RNA-bacteriophage AP205.
[0141] In a further preferred embodiment of the present invention,
the recombinant proteins comprise, or alternatively consist
essentially of, or alternatively consist of coat proteins of RNA
phages.
[0142] RNA-phage coat proteins forming capsids or VLPs, or
fragments of the bacteriophage coat proteins compatible with
self-assembly into a capsid or a VLP, are, therefore, further
preferred embodiments of the present invention. Bacteriophage
Q.beta. coat proteins, for example, can be expressed recombinantly
in E. coli. Further, upon such expression these proteins
spontaneously form capsids. Additionally, these capsids form a
structure with an inherent repetitive organization.
[0143] Specific preferred examples of bacteriophage coat proteins
which can be used to prepare compositions of the invention include
the coat proteins of RNA bacteriophages such as bacteriophage
Q.beta. (SEQ ID NO:4; PIR Database, Accession No. VCBPQ.beta.
referring to Q.beta. CP and SEQ ID NO: 5; Accession No. AAA 16663
referring to Q.beta. A1 protein), bacteriophage R17 (SEQ ID NO:6;
FIR Accession No. VCBPR7), bacteriophage fr (SEQ ID NO:7; PIR
Accession No. VCBPFR), bacteriophage GA (SEQ ID NO:8; GenBank
Accession No. NP-040754), bacteriophage SP (SEQ ID NO:9; GenBank
Accession No. CAA30374 referring to SP CP and SEQ ID NO: 10;
Accession No. NP 695026 referring to SP A1 protein), bacteriophage
MS2 (SEQ ID NO: 11; PIR Accession No. VCBPM2), bacteriophage M11
(SEQ ID NO: 12; GenBank Accession No. AAC06250), bacteriophage MX1
(SEQ ID NO: 13; GenBank Accession No. AAC14699), bacteriophage NL95
(SEQ ID NO: 14; GenBank Accession No. AAC14704), bacteriophage f2
(SEQ ID NO: 15; GenBank. Accession No. P03611), bacteriophage PP7
(SEQ ID NO: 16), and bacteriophage AP205 (SEQ ID NO: 28).
Furthermore, the A1 protein of bacteriophage Q.beta. (SEQ ID NO: 5)
or C-terminal truncated forms missing as much as 100, 150 or 180
amino acids from its C-terminus may be incorporated in a capsid
assembly of Q.beta. coat proteins. Generally, the percentage of
Q.beta. A1 protein relative to Q.beta. CP in the capsid assembly
will be limited, in order to ensure capsid formation.
[0144] Q.beta. coat protein has also been found to self-assemble
into capsids when expressed in E. coli (Kozlovska T M. et al., GENE
137: 133-137 (1993)). The obtained capsids or virus-like particles
showed an icosahedral phage-like capsid structure with a diameter
of 25 nm and T=3 quasi symmetry. Further, the crystal structure of
phage Q.beta. has been solved. The capsid contains 180 copies of
the coat protein, which are linked in covalent pentamers and
hexamers by disulfide bridges (Golmohammadi, R. et al. Structure 4:
543-5554 (1996)) leading to a remarkable stability of the capsid of
Q.beta. coat protein. Capsids or VLPs made from recombinant Q.beta.
coat protein may contain, however, subunits not linked via
disulfide links to other subunits within the capsid, or
incompletely linked. However, typically more than about 80% of the
subunits are linked via disulfide bridges to each other within the
VLP. Thus, upon loading recombinant Q.beta. capsid on non-reducing
SDS-PAGE, bands corresponding to monomeric Q.beta. coat protein as
well as bands corresponding to the hexamer or pentamer of Q.beta.
coat protein are visible. Incompletely disulfide-linked subunits
could appear as dimer, trimer or even tetramer bands in
non-reducing SDS-PAGE. Q.beta. capsid protein also shows unusual
resistance to organic solvents and denaturing agents. Surprisingly,
we have observed that DMSO and acetonitrile concentrations as high
as 30%, and Guanidinium concentrations as high as 1 M do not affect
the stability of the capsid. The high stability of the capsid of
Q.beta. coat protein is an advantageous feature, in particular, for
its use in immunization and vaccination of mammals and humans in
accordance of the present invention.
[0145] Upon expression in E. coli, the N-terminal methionine of
Q.beta. coat protein is usually-removed, as we observed by
N-terminal Edman sequencing as described in Stoll, E. et al. J.
Biol. Chem. 232: 990-993 (1977). VLP composed from Q.beta. coat
proteins where the N-terminal methionine has not been removed, or
VLPs comprising a mixture of Q.beta. coat proteins where the
N-terminal methionine is either cleaved or present are also within
the scope of the present invention.
[0146] Further preferred virus-like particles of RNA-phages, in
particular of Q.beta., accordance of this invention are disclosed
in WO 02/056901 the disclosure of which is herewith incorporated by
reference in its entirety.
[0147] Further RNA phage coat proteins have also been shown to
self-assemble upon expression in a bacterial host (Kastelein, R. A.
et al., Gene 23: 245-254 (1983), Kozlovskaya, T M. et al., Dokl.
Akad. Nauk SSSR 287: 452-455 (1986). Adhin, M R. et al., Virology
170: 238-242 (1989), Ni, C Z., et al., Protein Sci. 5: 2485-2493
(1996), Priano, C. et al., J. Mol. Biol. 249: 283-297 (1995)). The
Q.beta. phage capsid contains, in addition to the coat protein, the
so called read-through protein A1 and the maturation protein A2. A1
is generated by suppression at the UGA stop codon and has a length
of 329 aa. The capsid of phage Q.beta. recombinant coat protein
used in the invention is devoid of the A2 lysis protein, and
contains RNA from the host. The coat protein of RNA phages is an
RNA binding protein, and interacts with the stem loop of the
ribosomal binding site of the replicase gene acting as a
translational repressor during the life cycle of the virus. The
sequence and structural elements of the interaction are known
(Witherall, G W. & Uhlenbeck, O C. Biochemistry 28: 71-76
(1989); Lim F. et al., J. Biol. Chem. 271: 31839-31845 (1996)). The
stem loop and RNA in general are known to be involved in the virus
assembly (Golmohammadi, R. et al., Structure 4: 543-5554
(1996)).
[0148] In a further preferred embodiment of the present invention,
the virus-like particle comprises, or alternatively consists
essentially of or alternatively consists of recombinant proteins,
or fragments thereof, of a RNA-phage, wherein the recombinant
proteins comprise, alternatively consist essentially of or
alternatively consist of mutant coat proteins of a RNA phage,
preferably of mutant coat proteins of the RNA phages mentioned
above. In another preferred embodiment, the mutant coat proteins of
the RNA phage have been modified by removal of at least one, or
alternatively at least two, lysine residue by way of substitution,
or by addition of at least one lysine residue by way of
substitution; alternatively, the mutant coat proteins of the RNA
phage have been modified by deletion of at least one, or
alternatively at least two, lysine residue, or by addition of at
least one lysine residue by way of insertion. The deletion,
substitution or addition of at least one lysine residue allows
varying the degree of coupling, i.e. the amount of A.beta.1-6
peptides per subunits of the VLP of the RNA-phages, in particular,
to match and tailor the requirements of the vaccine.
[0149] In another preferred embodiment, the virus-like particle
comprises, or alternatively consists essentially of or
alternatively consists of recombinant proteins, or fragments
thereof, of the RNA-bacteriophage Q.beta., wherein the recombinant
proteins comprise, or alternatively consist essentially of, or
alternatively consist of coat proteins having an amino acid
sequence of SEQ ID NO:4, or a mixture of coat proteins having amino
acid sequences of SEQ ID NO:4 and of SEQ ID NO: 5 or mutants of SEQ
ID NO: 5 and wherein the N-terminal methionine is preferably
cleaved.
[0150] In a further preferred embodiment of the present invention,
the virus-like particle comprises, consists essentially of or
alternatively consists of recombinant proteins of Q.beta., or
fragments thereof wherein the recombinant proteins comprise, or
alternatively consist essentially of, or alternatively consist of
mutant Q.beta. coat proteins. In another preferred embodiment,
these mutant coat proteins have been modified by removal of at
least one lysine residue by way of substitution, or by addition of
at least one lysine residue by way of substitution. Alternatively,
these mutant coat proteins have been modified by deletion of at
least one lysine residue, or by addition of at least one lysine
residue: by way of insertion.
[0151] Four lysine residues are exposed on the surface of the
capsid of Q.beta. coat protein. Q.beta. mutants, for which exposed
lysine residues are replaced by arginines can also be used for the
present invention. The following Q.beta. coat protein mutants and
mutant Q.beta. VLPs can, thus, be used in the practice of the
invention.: "Q.beta.-240" (Lys13-Arg; SEQ ID NO:17), "Q.beta.-243"
(Asn 10-Lys; SEQ ID NO:18), "Q.beta.-250" (Lys 2-Arg, Lys13-Arg;
SEQ ID NO: 19), "Q.beta.-251" (SEQ ID NO:20) and "Q.beta.-259" (Lys
2-Arg, Lys16-Arg: SEQ ID NO:21). Thus, in further preferred
embodiment of the present invention, the virus-like particle
comprises, consists essentially of or alternatively consists of
recombinant proteins of mutant Q.beta. coat proteins, which
comprise proteins having an amino acid sequence selected from the
group of a) the amino acid sequence of SEQ ID NO: 17; b) the amino
acid sequence of SEQ ID NO:18; c) the amino acid sequence of SEQ ID
NO: 19; d) the amino acid sequence of SEQ ID NO:20; and e) the
amino acid sequence of SEQ ID NO: 2. The construction, expression
and purification of the above indicated Q.beta. coat proteins,
mutant Q.beta. coat protein VLPs and capsids,. respectively, are
described in WO 02/056905. In particular is hereby referred to
Example 18 of above mentioned application.
[0152] In a further preferred embodiment of the present invention,
the virus-like particle comprises, or alternatively consists
essentially of, or alternatively consists of recombinant proteins
of Q.beta., or fragments thereof, wherein the recombinant proteins
comprise, consist essentially of or alternatively consist of a
mixture of either one of the foregoing Q.beta. mutants and the
corresponding A1 protein.
[0153] In a further preferred embodiment of the present invention,
the virus-like particle comprises, or alternatively essentially
consists of, or alternatively consists of recombinant proteins, or
fragments thereof, of RNA-phage AP205.
[0154] The AP205 genome consists of a maturation protein, a coat
protein, a replicase and two open reading frames not present in
related phages; a lysis gene and an open reading frame playing a
role in the translation of the maturation gene (Klovins, J., et
al., J. Gen. Virol. 83: 1523-33 (2002)). AP205 coat protein can be
expressed from plasmid pAP283-58 (SEQ ID NO: 27), which is a
derivative of pQb10 (Kozlovska, T. M., et al., Gene 137: 133-37
(1993)), and which contains an AP205 ribosomal binding site.
Alternatively, AP205 coat protein may be cloned into pQb185,
downstream of the ribosomal binding site present in the vector.
Both approaches lead to expression of the protein and formation of
capsids as described in the co-pending US provisional patent
application with the title "Molecular Antigen Arrays" (Atty. Docket
No. 1700.0310000) and having been filed on Jul. 17, 2002, which is
incorporated by reference in its entirety. Vectors pQb10 and pQb185
are vectors derived from pGEM vector, and expression of the cloned
genes in these vectors is controlled by the trp promoter
(Kozlovska, T. M. et al., Gene 137: 133-37 (1993)). Plasmid
pAP283-58 (SEQ ID NO:27) comprises a putative AP205 ribosomal
binding site in the following sequence, which is downstream of the
XbaI site, and immediately upstream of the ATG start codon of the
AP205 coat protein: tctagaATTTTCTGCGCACCCAT
CCCGGGTGGCGCCCAAAGTGAGGAAAATCACatg (SEQ ID NO:57). The vector
pQb185 comprises a Shine Delagarno sequence downstream from the
XbaI site and upstream of the start codon
(tctagaTTAACCCAACGCGTAGGAG TCAGGCCatg (SEQ ID NO:58). Shine
Delagarno sequence underlined).
[0155] In a further preferred embodiment of the present invention,
the virus-like particle comprises, or alternatively essentially
consists of, or alternatively consists of recombinant coat
proteins, or fragments thereof, of the RNA-phage AP205.
[0156] This preferred embodiment of the present invention, thus,
comprises AP205 coat proteins that form capsids. Such proteins are
recombinantly expressed, or prepared from natural sources, AP205
coat proteins produced in bacteria spontaneously form capsids, as
evidenced by Electron Microscopy (EM) and immunodiffusion. The
structural properties of the capsid formed by the AP205 coat
protein (SEQ ID NO: 28) and those formed by the coat protein of the
AP205 RNA phage are nearly indistinguishable when seen in EM. AP205
VLPs are highly immunogenic, and can be linked with antigens and/or
antigenic determinants to generate vaccine constructs displaying
the antigens and/or antigenic determinants oriented in a repetitive
manner. High titers are elicited against the so displayed antigens
showing that bound antigens and/or antigenic determinants are
accessible for interacting with antibody molecules and are
immunogenic.
[0157] In a further preferred embodiment of the present invention,
the virus-like particle comprises, or alternatively essentially
consists of or alternatively consists of recombinant mutant coat
proteins, or fragments thereof, of the RNA-phage AP205.
[0158] Assembly-competent mutant forms of AP205 VLPs, including
AP205 coat protein with the substitution of proline at amino acid 5
to threonine (SEQ ID NO: 29), may also be used in the practice of
the invention and leads to a further preferred embodiment of the
invention. These VLPs, AP205 VLPs derived from natural sources, or
AP205 viral particles, may be bound to antigens to produce ordered
repetitive arrays of the antigens in accordance with the present
invention.
[0159] AP205 P5-T mutant coat protein can be expressed from plasmid
pAP281-32 (SEQ ID No. 30), which is derived directly from pQb185,
and which contains the mutant AP205 coat protein gene instead of
the Q.beta. coat protein gene. Vectors for expression of the AP205
coat protein are transfected info E. coli for expression of the
AP205 coat protein.
[0160] Methods for expression of the coat protein and the mutant
coat protein, respectively, leading to the self-assembly into VLPs
are described in Example 1. Suitable E. coli strains include, but
are not limited to, E. coli K802, M 109, RR1. Suitable vectors and
strains and combinations thereof can be identified by testing
expression of the coat protein and mutant coat protein,
respectively, by SDS-PAGE and capsid formation and assembly by
optionally first purifying the capsids by gel filtration and
subsequently testing them in an immunodiffusion assay (Ouchterlony
test) or Electron Microscopy (Kozlovska, T. M., et al., Gene 137:
133-37 (1993)).
[0161] AP205 coat proteins expressed from the vectors pAP283-58 and
pAP281-32 may be devoid of the initial Methionine ammo-acid, due to
processing in the cytoplasm of E. coli. Cleaved, uncleaved forms of
AP205 VLP or mixtures thereof are further preferred embodiments of
the invention.
[0162] In a further preferred embodiment of the present invention,
the virus-like particle comprises, or alternatively essentially
consists of, or alternatively consists of a mixture of recombinant
coat proteins, or fragments thereof, of the RNA-phage AP205 and of
recombinant mutant coat proteins, or fragments thereof, of the
RNA-phage AP205.
[0163] In a further preferred embodiment of the present invention,
the virus-like particle comprises, or alternatively essentially
consists of, or alternatively consists of fragments of recombinant
coat proteins or recombinant mutant coat proteins of the RNA-phage
AP205.
[0164] Recombinant AP205 coat protein fragments capable of
assembling into a VLP and a capsid, respectively are also useful in
the practice of the invention. These fragments may be generated by
deletion, either internally or at the termini of the coat protein
and mutant coat protein, respectively. Insertions in the coat
protein and mutant coat protein sequence or fusions of antigen
sequences to the coat protein and mutant coat protein sequence, and
compatible with assembly into a VLP, are further embodiments of the
invention and lead to chimeric AP205 coat proteins, and particles,
respectively. The outcome of insertions, deletions and fusions to
the coat protein sequence and whether if is compatible with
assembly into a VLP can be determined by electron microscopy.
[0165] The particles formed by the AP205 coat protein, coat,
protein fragments and chimeric coat proteins described above, can
be isolated in pure form by a combination of fractionation steps by
precipitation and of purification steps by gel filtration using
e.g. Sepharose CL-4B, Sepharose CL-2B, Sepharose CL-6B columns and
combinations thereof. Other methods of isolating virus-like
particles are known in the art, and may be used to isolate the
virus-like particles (VLPs) of bacteriophage AP205. For example,
the use of ulfracentrifugation to isolate VLPs of the yeast
retrotransposon Ty is described in U.S. Pat. No. 4,918,166, which
is incorporated by reference herein in its entirety.
[0166] The crystal structure of several RNA bacteriophages has been
determined (Golmohammadi, R. et al., Structure 4: 543-554 (1996)).
Using such information, surface exposed residues can be identified
and, thus, RNA-phage coat proteins can be modified such that one or
more reactive amino acid residues can be inserted by way of
insertion or substitution. As a consequence, those modified forms
of bacteriophage coat proteins can also be used for the present
invention. Thus, variants of proteins which form capsids or
capsid-like structures (e.g., coat proteins of bacteriophage
Q.beta., bacteriophage R17, bacteriophage fr, bacteriophage GA,
bacteriophage SP, bacteriophage AP205, and bacteriophage MS2) can
also be used to prepare compositions of the present invention.
[0167] Although the sequence of the variants proteins discussed
above will differ from their wild-type counterparts, these variant
proteins will generally retain the ability to form capsids or
capsid-like structures. Thus, the invention further includes
compositions and vaccine compositions, respectively, which further
includes variants of proteins which form capsids or capsid-like
structures, as well as methods for preparing such compositions and
vaccine compositions, respectively, individual protein subunits
used to prepare such compositions, and nucleic acid molecules which
encode these protein subunits. Thus, included within the scope of
the invention are variant forms of wild-type proteins which form
capsids or capsid-like structures and retain the ability to
associate and form capsids or capsid-like structures.
[0168] As a result, the invention further includes compositions and
vaccine compositions, respectively, comprising proteins, which
comprise, or alternatively consist essentially of, or alternatively
consist of amino acid sequences which are at least 80%, 85%, 90%,
95%, 97%, or 99% identical to wild-type proteins which form ordered
arrays and have an inherent repetitive structure, respectively.
[0169] Further included within the scope of the invention are
nucleic acid molecules which encode proteins used to prepare
compositions of the present invention.
[0170] In other embodiments, the invention further includes
compositions comprising proteins, which comprise, or alternatively
consist essentially of, or alternatively consist of amino acid
sequences which are at least 80%, 85%, 90%, 95%, 97%, or 99%
identical to any of the amino acid sequences shown in SEQ ID
NOs:4-21.
[0171] Proteins suitable for use in the present invention also
include C-terminal truncation mutants of proteins which form
capsids or capsid-like structures, or VLPs. Specific examples of
such truncation mutants include proteins having an amino acid
sequence shown in any of SEQ ID NOS:4-21 where 1, 2, 5, 7, 9, 10,
12, 14, 15, or 17 amino acids have been removed from the
C-terminus. Typically, theses C-terminal truncation mutants will
retain the ability to form capsids or capsid-like structures.
[0172] Further proteins suitable for use in the present invention
also include N-terminal truncation mutants of proteins which form
capsids or capsid-like structures. Specific examples of such
truncation mutants include proteins having an amino acid sequence
shown in any of SEQ ID NOs:4-21 where 1, 2, 5, 7, 9, 10, 12, 14,
15, or 17 amino acids have been removed from the N-terminus.
Typically, these N-terminal truncation mutants, will retain the
ability to form capsids or capsid-like structures.
[0173] Additional proteins suitable for use in the present
invention include N- and C-terminal truncation mutants which form
capsids or capsid-like structures. Suitable truncation mutants
include proteins having an amino acid sequence shown in any of SEQ
ID NOs:4-21 where 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids
have been removed from the N-terminus and 1, 2, 5, 7, 9, 10, 12,
14, 15, or 17 amino acids have been removed from the C-terminus,
Typically, these N-terminal and C-terminal truncation mutants will
retain the ability to form capsids or capsid-like structures.
[0174] The invention further includes compositions comprising
proteins which comprise, or alternatively consist essentially of,
or alternatively consist of, amino acid sequences which are at
least 80%, 85%, 90%, 95%, 97%, or 99% identical to the above
described truncation mutants.
[0175] The invention thus includes compositions and vaccine
compositions prepared from proteins which form capsids or VLPs,
methods for preparing these compositions from individual protein
subunits and VLPs or capsids, methods for preparing these
individual protein subunits, nucleic acid molecules which encode
these subunits, and methods for vaccinating and/or eliciting
immunological responses in individuals using these compositions of
the present invention.
[0176] As previously stated, the invention includes virus-like
particles or recombinant forms thereof. In one further preferred
embodiment, the particles used in compositions of the invention are
composed of a Hepatitis B core protein (HBcAg) or a fragment of a
HBcAg. In a further embodiment, the particles used in compositions
of the invention are composed of a Hepatitis B core protein (HBcAg)
or a fragment of a HBcAg protein, which has been modified to either
eliminate or reduce the number of free cysteine residues. Zhou et
at (J. Virol. 66: 5393-5398 (1992)) demonstrated that HBcAgs which
have been modified to remove the naturally resident cysteine
residues retain the ability to associate and form capsids. Thus,
VLPs suitable for use in compositions of the invention include
those comprising modified HBcAgs, or fragments thereof, in which
one or more of the naturally resident cysteine residues have been
either deleted or substituted with another amino acid residue
(e.g., a serine residue).
[0177] The HBcAg is a protein generated by the processing of a
Hepatitis B core antigen precursor protein. A number of isotypes of
the HBcAg have been identified and their amino acids sequences are
readily available to those skilled in the art. In most instances,
compositions and vaccine compositions, respectively, of the
invention will be prepared using the processed form of a HBcAg
(i.e., a HBcAg from which the N-terminal leader sequence of the
Hepatitis B core antigen precursor protein have been removed).
[0178] Further, when HBcAgs are produced under conditions where
processing will not occur, the HBcAgs will generally be expressed
in "processed" form. For example, when an E. coli expression system
directing expression of the protein to the cytoplasm, is used to
produce HBcAgs of the invention, these proteins will generally be
expressed such that the N-terminal leader sequence of the Hepatitis
B core antigen precursor protein is not present.
[0179] The preparation of Hepatitis B virus-like particles, which
can be used for the present invention, is disclosed, for example,
in WO 00/32227, and hereby in particular in Examples 17 to 19 and
21 to 24, as well as in WO 81/85208, and hereby in particular in
Examples 1 to 19, 21 to 24, 31 and 41, and in WO 02/056905. For the
latter application, it is in particular referred to Example 23, 24,
31 and 51. All three documents are explicitly incorporated herein
by reference.
[0180] The present invention also includes HBcAg variants which
have been modified to delete or substitute one or more additional
cysteine residues. It is known in the art that free cysteine
residues can be involved in a number of chemical side reactions.
These side reactions include disulfide exchanges, reaction with
chemical substances or metabolites that are, for example, injected
or formed in a combination therapy with other substances, or direct
oxidation and reaction with nucleotides upon exposure to UV light.
Toxic adducts could thus be generated, especially considering the
fact that HBcAgs have a strong tendency to bind nucleic acids. The
toxic adducts would thus be distributed between a multiplicity of
species, which individually may each be present at low
concentration, but reach toxic levels when together.
[0181] In view of the above, one advantage to the use of HBcAgs in
vaccine compositions which have been modified to remove naturally
resident cysteine residues is that sites to which toxic species,
can bind when antigens or antigenic determinants are attached would
be reduced in number of eliminated altogether.
[0182] A number of naturally occurring HBcAg variants suitable for
use in the practice of the present invention have been identified.
Yuan et al., (J. Virol. 73: 10122-10128 (1999)), for example,
describe variants in which the isoleucine residue at position
corresponding to position 97 in SEQ ID NO:22 is replaced with
either a leucine residue or phenylalanine residue. The amino acid
sequences of a number of HBcAg variants, as well as several.
Hepatitis B core antigen precursor variants, are disclosed in
GenBank reports AAF121240, AF121239, X85297, X02496, X85305,
X85303, AF51735, X85259, X85286, X85260, X85317, X85298, AF043593,
M20706, X85295, X80925, X85284, X85275, X72702, X85291, X65258,
X85302, M32138, X85293, X85315, U95551, X85256, X85316, X85296,
AB033559, X59795, X85299, X85307, X65257, X85311, X85301 (SEQ ID
NO:23), X85314, X85287, X85272, X85319, AB010289, X85285, AB010289,
AF121242, M90520 (SEQ ID NO:24), P03153, AF110999, and M95589, the
disclosures of each of which are incorporated herein by reference.
The sequences of the hereinabove mentioned Hepatitis B core antigen
precursor variants are further disclosed in WO 01/85208 in SEQ ID
NOs: 89-138. These HBcAg variants differ in amino acid sequence at
a number of positions, including amino acid residues which
corresponds to the amino acid residues located at positions: 12,
13, 21, 22, 24, 29, 32, 33, 35, 38, 40, 42, 44, 45, 49, 51, 57, 58,
59,64, 66, 67, 69, 74, 77, 80, 81, 87, 92, 93, 97, 98, 100, 103,
105, 106, 109, 113, 116, 121, 126, 130, 133, 135, 141, 147, 149,
157, 176, 178, 182 and 183 in SEQ ID NO:25. Further HBcAg variants
suitable for use in the compositions of the invention, and which
may be further modified according to the disclosure of this
specification are described in WO 007198333, WO 00/177158 and WO
00/214478.
[0183] As noted above, generally processed HBcAgs (i.e., those
which lack leader sequences) will be used in the compositions and
vaccine compositions, respectively, of the invention. The present
invention includes vaccine compositions, as well as methods for
using these compositions, which employ the above described variant
HBcAgs.
[0184] Whether the amino acid sequence of a polypeptide has an
amino acid sequence that is at least 80%, 85%, 90%, 95%, 97% or 99%
identical to one of the above wild-type amino acid sequences, or a
subportion thereof, can be determined conventionally using known
computer program's such the Bestfit program. When using Bestfit or
any other sequence alignment program to determine whether a
particular sequence is, for instance, 95% identical to a reference
amino acid sequence, the parameters are set such that the
percentage of identify is calculated over the full length of the
reference amino acid sequence and that gaps in homology of up to 5%
of the total number of amino acid residues in the reference
sequence are allowed.
[0185] The amino acid sequences of the hereinabove mentioned HBcAg
variants and precursors are relatively similar to each other. Thus,
reference to an amino acid residue of a HBcAg variant located at a
position which corresponds to a particular position in SEQ ID
NO:25, refers to the amino acid residue which is present at that
position in the amino acid sequence shown in SEQ ID NO:25. The
homology between these HBcAg variants is for the most part high
enough among Hepatitis B viruses that infect mammals so that one
skilled in the art would have little difficulty reviewing both the
amino acid sequence shown in SEQ ID NO:25 and that of a particular
HBcAg variant and identifying "corresponding" amino acid residues.
Furthermore, the HBcAg amino acid sequence shown in SEQ ID NO:24,
which shows the amino acid sequence of a HBcAg derived from a virus
which infect woodchucks, has enough homology to the HBcAg having
the amino acid sequence shown in SEQ ID NO:33 that it is readily
apparent that a three amino acid residue insert is present in SEQ
ID NO:25 between amino acid residues 155 and 156 of SEQ ID
NO:25.
[0186] The invention also includes vaccine compositions which
comprise HBcAg variants of Hepatitis B viruses which infect birds,
as wells as vaccine compositions which comprise fragments of these
HBcAg variants. For these HBcAg variants one, two, three or more of
the cysteine residues naturally present in these polypeptides could
be either substituted with another amino acid residue or deleted
prior to their inclusion in vaccine compositions of the
invention.
[0187] As discussed above, the elimination of free cysteine
residues reduces the number of sites where toxic components can
bind to the HBcAg, and also eliminates sites where cross-linking of
lysine and cysteine residues of the same or of neighboring HBcAg
molecules can occur. Therefore, in another embodiment of the
present invention, one or more cysteine residues of the Hepatitis B
virus capsid protein have been either deleted or substituted with
another amino acid residue.
[0188] In other embodiments, compositions and vaccine compositions,
respectively, of the invention will contain HBcAgs from which the
C-terminal region (e.g., amino acid residues 145-185 or 150-185 of
SEQ ID NO: 25) has been removed. Thus, additional modified HBcAgs
suitable for use in the practice of the present invention include
C-terminal truncation mutants. Suitable truncation mutants include
HBcAgs where 1, 5, 10, 15, 20, 25, 30, 34, 35, amino acids have
been removed from the C-terminus.
[0189] HBcAgs suitable for use in the practice of the present
invention also include N-terminal truncation mutants. Suitable
truncation mutants include modified HBcAgs where 1, 2, 5, 7, 9, 10,
12, 14, 15, or 17 amino acids have been removed from the
N-terminus.
[0190] Further HBcAgs suitable for use in the practice of the
present invention include N- and C-terminal truncation mutants.
Suitable truncation mutants include HBcAgs where 1, 2, 5, 7, 9, 10,
12, 14, 15, and 17 amino acids have been removed from the
N-terminus and 1, 5, 10, 15, 20, 25, 30, 34, 35 amino acids have
been removed from the C-terminus.
[0191] The invention further includes compositions and vaccine
compositions, respectively, comprising HBcAg polypeptides
comprising, or alternatively essentially consisting of, or
alternatively consisting of, amino acid sequences which are at
least 80%, 85%, 90%, 95%, 97%, or 99% identical to the above
described truncation mutants.
[0192] In certain embodiments of the invention, a lysine residue is
introduced into a HBcAg polypeptide, to mediate the binding of the
A.beta.1-6 peptide to the VLP of HBcAg. In preferred embodiments,
compositions of the invention are prepared using a HBcAg
comprising, or alternatively consisting of, amino acids 1-144, or
1-149, 1-185 of SEQ ID NO:25, which is modified so that the amino
acids corresponding to positions 79 and 80 are replaced with a
peptide having the amino acid sequence of Gly-Gly-Lys-Gly-Gly (SEQ
ID NO:33) resulting in the HBcAg polypeptide having the sequence
shown in SEQ ID NO: 26. These compositions are particularly useful
in those embodiments where an antigenic determinant is coupled to a
VLP of HBcAg. In further preferred embodiments, the cysteine
residues at positions 48 and 107 of SEQ ID NO:25 are mutated to
serine. The invention further includes compositions comprising the
corresponding polypeptides having amino acid sequences shown in any
of the hereinabove mentioned Hepatitis B core antigen precursor
variants which also have above noted amino acid alterations.
Further included within the scope of the invention are additional
HBcAg variants which are capable of associating to form a capsid or
VLP and have the above noted amino acid alterations. Thus, the
invention further includes compositions and vaccine compositions,
respectively, comprising HBcAg polypeptides which comprise, or
alternatively consist of, amino acid sequences which are at least
80%, 85%, 90%, 95%, 97% or 99% identical to any of the wild-type
amino acid sequences, and forms of these proteins which have been
processed, where appropriate, to remove the N-terminal leader
sequence and modified with above noted alterations.
[0193] Compositions or vaccine compositions of the invention may
comprise mixtures of different HBcAgs. Thus, these vaccine
compositions may be composed of HBcAgs which differ in amino acid
sequence. For example, vaccine compositions could be prepared
comprising a "wild-type" HBcAg and a modified HBcAg in which one or
more amino acid residues have been altered (e.g., deleted, inserted
or substituted). Further, preferred vaccine compositions of the
invention are those which present highly ordered and repetitive
antigen arrays, wherein the antigen is a A.beta.1-6 peptide.
[0194] In a further preferred embodiment of the present invention,
the at least one A.beta.1-6 peptide is bound to said virus-like
particle and core particle, respectively, by at least one covalent
bond. Preferably, the least one A.beta.1-6 peptide is bound to the
virus-like particle and core particle, respectively, by at least
one covalent bond, said covalent bond being a non-peptide bond
leading to a A.beta.1-6 peptide array and A.beta.1-6 peptide -VLP
conjugate, respectively. This A.beta.1-6 peptide array and
conjugate, respectively, has typically and preferably a repetitive
and ordered structure since the at least one A.beta.1-6 peptide is
bound to the VLP and core particle, respectively, in an oriented
manner. The formation of a repetitive and ordered A.beta.1-6
peptide -VLP array and conjugate, respectively, is ensured by an
oriented and directed as well as defined binding and attachment,
respectively, of the at least one A.beta.1-6 peptide to the VLP and
core particle, respectively, as will become apparent in the
following. Furthermore, the typical inherent highly repetitive and
organized structure of the VLPs and core particles, respectively,
advantageously contributes to the display of the A.beta.1-6 peptide
in a highly ordered and repetitive fashion leading to a highly
organized and repetitive A.beta.1-6 peptide-VLP array and
conjugate, respectively.
[0195] Therefore, the preferred inventive conjugates and arrays,
respectively, differ from prior art conjugates in their highly
organized structure, dimensions, and in the repetitiveness of the
antigen on the surface of the array. The preferred embodiment of
this invention, furthermore, allows expression of the particle in
an expression host guaranteeing proper folding and assembly of the
VLP, to which the antigen, i.e. the A.beta.1-6 peptide, is then
further coupled
[0196] The present invention discloses methods of binding of
A.beta.1-6 peptide to VLPs. As indicated, in one aspect of the
invention, the A.beta.1-6 peptide is bound to the VLP by way of
chemical cross-linking, typically and preferably by using a
heterobifunctional cross-linker. Several hetero-bifunctional
cross-linkers are known to the art, in preferred embodiments, the
hetero-bifunctional cross-linker contains a functional group which
can react with preferred first attachment sites, i.e. with the
side-chain amino group of lysine residues of the VLP or at least
one VLP subunit, and a further functional group which can react
with a preferred second attachment site, i.e. a cysteine residue
fused to the A.beta.1-6 peptide and optionally also made available
for reaction by reduction. The first step of the procedure,
typically called the derivatization, is the reaction of the VLP
with the cross-linker. The product of this reaction is an activated
VLP, also called activated carrier. In the second step, unreacted
cross-linker is removed using usual methods such as gel filtration
or dialysis. In the third step, the A.beta.1-6 peptide is reacted
with the activated VLP, and this step is typically called the
coupling step. Unreacted A.beta.1-6 peptide may be optionally
removed in a fourth step, for example by dialysis. Several
hetero-bifunctional cross-linkers are known to the art. These
include, the preferred cross-linkers SMPH (Pierce), Sulfo-MBS,
Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, SVSB,
SIA and other cross-linkers available for example from the Pierce
Chemical Company (Rockford, Ill., USA), and having one functional
group reactive towards amino groups and one functional group
reactive towards cysteine residues. The above mentioned
cross-linkers all lead to formation of a thioether linkage. Another
class of cross-linkers suitable in the practice of the invention is
characterized by the introduction of a disulfide linkage between
the A.beta.1-6 peptide and the VLP upon coupling. Preferred
cross-linkers belonging to this class include for example SPDP and
Sulfo-LC-SPDP (Pierce). The extent of derivatization of the VLP
with cross-linker can be influenced by varying experimental
conditions such as the concentration of each of the reaction
partners, the excess of one reagent over the other, the pH, the
temperature and the ionic strength. The degree of coupling, i.e.
the amount of A.beta.1-6 peptides per subunits of the VLP can be
adjusted by varying the experimental conditions described above to
match the requirements of the vaccine.
[0197] A particularly favored method of binding of A.beta.1-6
peptides to the VLP, is the linking of a lysine residue on the
surface of the VLP with a cysteine residue on the A.beta.1-6
peptide. In some embodiments, fusion of an amino acid linker
containing a cysteine residue, as a second attachment site or as a
part thereof, to A.beta.1-6 for coupling to the VLP may be
required.
[0198] In general, flexible amino acid linkers are favored.
Examples of the amino acid linker are selected from the group
consisting of: (a) CGG; (b) N-terminal gamma 1-linker; (c)
N-terminal gamma 3-linker; (d) Ig hinge regions; (e) N-terminal
glycine linkers; (f) (G).sub.kC(G).sub.n with n=0-12 and k=0-5 (SEQ
ID NO: 34): (g) N-terminal glycine-serine linkers; (h)
(G).sub.kC(G).sub.m(S).sub.l(GGGGS).sub.n with n=0-3, k=0-5,
m=0-10, l=0-2 (SEQ ID NO: 35); (i) GGC; (k) GGC-NH2; (l) C-terminal
gamma 1-linker; (m) C-terminal gamma 3-linker; (n) C-terminal
glycine linkers; (o) (G).sub.nC(G).sub.k with n=0-12 and k=0-5 (SEQ
ID NO: 36); (p) C-terminal glycine-serine linkers; (q)
(G).sub.m(S).sub.l(GGGGS).sub.n(G).sub.oC(G).sub.k with n=0-3,
k=0-5, m=0-10, l=0-2, and o=0-8 (SEQ ID NO: 37).
[0199] Further examples of amino acid linkers are the hinge region
of Immunoglobulins, glycine serine linkers (GGGGS).sub.n (SEQ ID
NO: 38), and glycine linkers (G).sub.n all further containing a
cysteine residue as second attachment site and optionally further
glycine residues. Typically preferred examples of said amino acid
linkers are N-terminal gamma1: CGDKTHTSPP (SEQ ID NO: 39);
C-terminal gamma 1: DKTHTSPPCG (SEQ ID NO: 40); N-terminal gamma 3:
CGGPKPSTPPGSSGGAP (SEQ ID NO: 41); C-terminal gamma 3:
PKPSTFPGSSGGAPGGCG (SEQ ID NO: 42); N-terminal glycine linker:
GCGGGG (SEQ ID NO: 43) and C-terminal glycine linker: GGGGCG (SEQ
ID NO: 44).
[0200] Other amino acid linkers particularly suitable in the
practice of the invention, when a hydrophobic A.beta. pepide is
bound to a VLP, are CGKKGG (SEQ ID NO: 46), or CGDEGG (SEQ ID NO:
31) for N-terminal linkers, or GGKKGC (SEQ ID NO: 45) and GGEDGC
(SEQ ID NO: 32), for the C-terminal linkers. For the C-terminal
linkers, the terminal cysteine is optionally C-terminally
amidated.
[0201] In preferred embodiments of the present invention, GGCG (SEQ
ID NO: 47), GGC or GGC-NH2 ("NH2" stands for amidation) linkers at
the C-terminus of the peptide or CCG at its N-terminus are
preferred as amino acid linkers. In general, glycine residues will
be inserted between bulky amino acids and the cysteine to be used
as second attachment site, to avoid potential steric hindrance of
the bulkier amino acid in the coupling reaction. In the most
preferred embodiment of the invention, the amino acid linker
GGC-NH2 is fused to the C-terminus of A.beta.1-6.
[0202] The cysteine residue present on the A.beta.1-6 peptide has
to be in its reduced state to react with the hetero-bifunctional
cross-linker on the activated VLP, that is a free cysteine or a
cysteine residue with a free sulfhydryl group has to be available.
In the instance where the cysteine residue to function as binding
site is in an oxidized form, for example if it is forming a
disulfide bridge, reduction of this disulfide bridge with e.g. DTT,
TCEP or .beta.-mercaptoethanol is required. Low concentrations of
reducing agent are compatible with coupling as described in WO
02/056905, higher concentrations inhibit the coupling reaction, as
a skilled artisan would know, in which case the reductand has to be
removed or its concentration decreased prior to coupling, e.g., by
dialysis, gel filtration or reverse phase HPLC.
[0203] Binding of the A.beta.1-6 peptide to the VLP by using a
hetero-bifunctional cross-linker according to the preferred methods
described above, allows coupling of A.beta.1-6 peptide to the VLP
in an oriented fashion. Other methods of binding the A.beta.1-6
peptide to the VLP include methods wherein the A.beta.1-6 peptide
is cross-linked to the VLP using the carbodiimide EDC, and NHS. In
further methods, the A.beta.1-6 peptide is attached to the VLP
using a homo-bifunctional cross-linker such as glutaraldehyde, DSG,
BM[PEO].sub.4, BS.sup.3, (Pierce Chemical Company, Rockford, Ill.,
USA) or other known homo-bifunctional cross-linkers with functional
groups reactive towards amine groups or carboxyl groups of the
VLP.
[0204] Other methods of binding the VLP to a A.beta.1-6 peptide
include methods where the VLP is biotinylated, and the A.beta.1-6
peptide expressed as a streptavidin-fusion protein, or methods
wherein both the A.beta.1-6 peptide and the VLP are biotinylated,
for example as described in WO 00/23955. In this case, the
A.beta.1-6 peptide may be first bound to streptavidin or avidin by
adjusting the ratio of A.beta.1-6 peptide to streptavidin such that
free binding sites are still available for binding of the VLP,
which is added in the next step. Alternatively, all components may
be mixed in a "one pot" reaction. Other ligand-receptor pairs,
where a soluble form of the receptor and of the ligand is
available, and are capable of being cross-linked to the VLP or the
A.beta.1-6 peptide, may be used as binding agents for binding
A.beta.1-6 peptide to the VLP. Alternatively, either the ligand or
the receptor may be fused to the A.beta.1-6 peptide, and so mediate
binding to the VLP chemically bound or fused either to the
receptor, or the ligand respectively. Fusion may also be effected
by insertion or substitution.
[0205] As already indicated, in a favored embodiment of the present
invention, the VLP is the VLP of a RNA phage, and in a more:
preferred embodiment, the VLP is the VLP of RNA phage Q.beta. coat
protein.
[0206] One or several antigen molecules, i.e. a A.beta.1-6 peptide,
can be attached to one submit of the capsid or VLP of RNA phages
coat proteins, preferably through the exposed lysine residues of
the VLP of RNA phages, if sterically allowable. A specific feature
of the VLP of the coat protein of RNA phages and in particular of
the Q.beta. coat protein VLP is thus the possibility to couple
several antigens per subpart. This allows for the generation of a
dense antigen array.
[0207] In a preferred embodiment of the invention, the binding and
attachment respectively, of the at least one A.beta.1-6 peptide to
the virus-like particle is by way of interaction and association,
respectively, between at least one first attachment site of the
virus-like particle and at least one second attachment of the
antigen or antigenic determinant.
[0208] VLPs or capsids of Q.beta. coat protein display a defined
number of lysine residues on their surface, with a defined topology
with three lysine residues pointing towards the interior of the
capsid and interacting with the RNA, and four other lysine residues
exposed to the exterior of the capsid. These defined properties
favor the attachment of antigens to the exterior of the particle,
rather than to the interior of the particle where the lysine
residues interact with RNA. VLPs of other RNA phage coat proteins
also have a defined number of lysine residues on their surface and
a defined topology of these lysine residues.
[0209] In further preferred embodiments of the present invention,
the first attachment site is a lysine residue and/or the second
attachment comprises sulfhydryl group or a cysteine residue. In a
very preferred embodiment of the present invention, the first
attachment site is a lysine residue and the second attachment is a
cysteine residue.
[0210] In very preferred embodiments of the invention, the
A.beta.1-6 peptide is bound via a cysteine residue, to lysine
residues of the VLP of RNA phage coat protein, and in particular to
the VLP of Q.beta. coat protein.
[0211] Another advantage of the VLPs derived from RNA phages is
their high expression yield in bacteria that allows production of
large quantities of material at affordable cost.
[0212] As indicated, the inventive conjugates and arrays,
respectively, differ from prior art conjugates in their highly
organized structure, dimensions, and in the repetitiveness of the
antigen on the surface of the array. Moreover, the use of the VLPs
as carriers allow the formation of robust antigen arrays and
conjugates, respectively, with variable antigen density. In
particular, the use of VLPs of RNA phages, and hereby in particular
the use of the VLP of RNA phage Q.beta. coat protein allows
achieving very high epitope density. In particular, a density of
more than 1.5 epitopes per subunit could be reached by coupling the
human A.beta.1-6 peptide to the VLP of Q.beta. coat protein. The
preparation of compositions of VLPs of RNA phage coat proteins with
a high epitope density can be effected using the teaching of this
application. In preferred embodiment of the invention, when a
A.beta.1-6 peptide is coupled to the VLP of Q6 coat protein, an
average number of A.beta.1-6 peptide per subunit of 0.5, 0.6, 0.7,
0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,
2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or higher is
preferred.
[0213] The second attachment site, as defined herein, may be either
naturally or non-naturally present with the antigen or the
antigenic determinant. In the case of the absence of a suitable
natural occurring second attachment site on the antigen or
antigenic determinant, such a, then non-natural second attachment
has to be engineered to the antigen.
[0214] As described above, four lysine residues are exposed on the
surface of the VLP Of Q.beta. coat protein. Typically these
residues are derivatized upon reaction with a cross-linker
molecule. In the instance where not all of the exposed lysine
residues can be coupled to an antigen, the lysine residues which
have reacted with the cross-linker are left with a cross-linker
molecule attached to the .epsilon.-amino group after the
derivatization step. This leads to disappearance of one or several
positive charges, which may be detrimental to the solubility and
stability of the VLP. By replacing some of the lysine residues with
arginines, as in the disclosed Q.beta. coat protein mutants
described below, we prevent the excessive disappearance of positive
charges since the arginine residues do not react with the
cross-linker. Moreover, replacement of lysine residues by arginines
may lead to more defined antigen arrays, as fewer sites are
available for reaction to the antigen.
[0215] Accordingly, exposed lysine residues were replaced by
arginines in the following Q.beta. coat protein mutants and mutant
Q.beta. VLPs disclosed in this application: Q.beta.-240 (Lys13-Arg:
SEQ ID NO:17), Q.beta.-250 (Lys 2-Arg, Lys13-Arg; SEQ ID NO: 19)
and Q.beta.-259 (Lys 2-Arg, Lys16-Arg; SEQ ID NO: 21). The
constructs were cloned, the proteins expressed, the VLPs purified
and used for coupling to peptide and protein antigens. Q.beta.-251;
(SEQ ID NO: 20 was also constructed, and guidance on how to
express, purify and couple the VLP of Q.beta.-251 coat protein can
be found throughout the application.
[0216] In a further embodiment, we disclose a Q.beta. mutant coat
protein with one additional lysine residue, suitable for obtaining
even higher density arrays of antigens, This mutant Q.beta. coat
protein, Q.beta.-243 (Asn 10-Lys; SEQ ID NO: 18), was cloned, the
protein expressed, and the capsid or VLP isolated and purified,
showing that introduction of the additional lysine residue is
compatible with self-assembly of the subunits to a capsid or VLP.
Thus, A.beta.1-6 peptide arrays, and conjugates, respectively, may
be prepared using VLP of Q.beta. coat protein mutants, A
particularly favored method of attachment of antigens to VLPs, and
in particular to VLPs of RNA phage coat proteins is the linking of
a lysine residue present on the surface of the VLP of RNA phage
coat proteins with a cysteine residue added to the antigen, i.e.
the A.beta.1-6 peptide, in order for a cysteine residue to be
effective as second attachment site, a sulfhydryl group must be
available for coupling. Thus, a cysteine residue has to be in its
reduced state, that is, a free cysteine or a cysteine residue with
a free sulfhydryl group has to be available. In the instant where
the cysteine residue to function as second attachment site is in an
oxidized form, for example if it is forming a disulfide bridge,
reduction of this disulfide bridge with e.g. DTP, TCEP or
.beta.-mercaptoethanol is required. The concentration of reductand,
and the molar excess of reductand over antigen has to be adjusted
for each antigen. A titration range, starting from concentrations
as low as 10 .mu.M or lower, up to 10 to 20 mM or higher reductand
if required is tested, and coupling of the antigen to the carrier
assessed. Although low concentrations of reductand are compatible
with the coupling reaction as described in WO 02/056905, higher
concentrations inhibit the coupling reaction, as a skilled artisan
would know, in which case the reductand has to be removed or its
concentration decreased, e.g. by dialysis, gel filtration or
reverse phase HPLC. Advantageously, the pH of the dialysis or
equilibration buffer is lower than 7, preferably 6. The
compatibility of the low pH buffer with antigen activity or
stability has to be tested.
[0217] Epitope density on the VLP of RNA phage coat proteins can be
modulated by the choice of cross-linker and other reaction
conditions. For example, the-cross-linkers Sulfo-GMBS and SMPH
typically allow reaching high epitope density. Derivatization is
positively influenced by high concentration of reactands, and
manipulation of the reaction conditions can be used to control the
number of antigens coupled to VLPs of RNA phage coat proteins, and
in particular to VLPs of Q.beta. coat protein.
[0218] Prior to the design of a non-natural second attachment site
the position at which it should be fused, inserted or generally
engineered has to be chosen. The selection of the position of the
second attachment site may, by way of example, be based on a
crystal structure of the antigen. Such a crystal structure of the
antigen may provide information on the availability of the C- or or
N-termini of the molecule (determined for example from their
accessibility to solvent), or on the exposure to solvent of
residues suitable for use as second attachment sites, such as
cysteine residues. Exposed disulfide bridges, as is the case for
Fab fragments, may also be a source of a second attachment site,
since they can be generally converted to single cysteine residues
through mild reduction, with e.g. 2-mercaptoethylamine, TCEP,
.beta.-mercaptoethanol or DTT. Mild reduction conditions not
affecting the immunogenicity of the antigen will be chosen. In
general, in the case where immunization with a self-antigen is
aiming at inhibiting the interaction of this self-antigen with its
natural ligands, the second attachment, site will be added such
that it allows generation of antibodies against the site of
interaction with the natural ligands. Thus, the location of the
second attachment site will be selected such that steric hindrance
from the second attachment site or any amino acid linker containing
the same is avoided. In further embodiments, an antibody response
directed at a site distinct from the interaction site of the
self-antigen with its natural ligand is desired. In such
embodiments, the second attachment site may be selected such that
it prevents generation of antibodies against the interaction site
of the self-antigen with its natural ligands.
[0219] Other criteria in selecting the position of the second
attachment site include the oligomerization state of the antigen,
the site of oligomerization, the presence of a cofactor, and the
availability of experimental evidence disclosing sites in the
antigen structure and sequence where modification of the antigen is
compatible with the function of the self-antigen, or with the
generation of antibodies recognizing the self-antigen.
[0220] In the most preferred embodiments, the A.beta.1-6 peptide
comprises a single second attachment site or a single reactive
attachment site capable of association with the first attachment
sites on the core particle and the VLPs or VLP subunits,
respectively. This ensures a defined and uniform binding and
association, respectively, of the at least one, but typically more
than one, preferably more than 10, 20, 40, 80, 120, 150, 180, 210,
240, 270, 300, 360, 400, 450 antigens to the core particle and VLP,
respectively. The provision of a single second attachment site or a
single reactive attachment site on the antigen, thus, ensures a
single and uniform type of binding and association, respectively
leading to a very highly ordered and repetitive array. For example,
if the binding and association, respectively, is effected by way of
a lysine- (as the first attachment site) and cysteine- (as a second
attachment site) interaction, it is ensured, in accordance with
this preferred embodiment of the invention, that only one cysteine
residue per antigen, independent whether this cysteine residue is
naturally or non-naturally present on the antigen, is capable of
binding and associating, respectively, with the VLP and the first
attachment site of the core particle, respectively.
[0221] In some embodiments, engineering of a second attachment site
onto the antigen require the fusion of an amino acid linker
containing an amino acid suitable as second attachment site
according to the disclosures of this invention. Therefore, in a
preferred embodiment of the present invention, an amino acid linker
is bound to the antigen or the antigenic determinant by way of at
least one covalent bond. Preferably, the amino acid linker
comprises, or alternatively consists of, the second attachment
site. In a further preferred embodiment, the amino acid linker
comprises a sulfhydryl group or a cysteine residue. In another
preferred embodiment, the amino acid linker is cysteine. Some
criteria of selection of the amino acid linker as well as further
preferred embodiments of the amino acid linker according to the
invention have already been mentioned above.
[0222] In a further preferred embodiment of the invention, the at
least one antigen or antigenic determinant i.e. the A.beta.1-6
peptide is fused to the virus-like particle. As outlined above, a
VLP is typically composed of at least one subunit assembling into a
VLP. Thus, in again a further preferred embodiment of the
invention, the antigen or antigenic determinant, preferably the at
least one A.beta.1-6 peptide is fused to at least one subunit of
the virus-like particle or of a protein capable of being
incorporated into a VLP generating, a chimeric
VLP-subunit-A.beta.1-6 peptide protein fusion.
[0223] Fusion of the A.beta.1-6 peptides can be effected by
insertion into the VLP subunit sequence, or by fusion to either the
N or C-terminus of the VLP-subunit or protein capable of being
incorporated into a VLP. Hereinafter, when referring to fusion
proteins of a peptide to a VLP subunit, the fusion to either ends
of the subunit sequence or internal insertion of the peptide within
the subunit sequence are encompassed.
[0224] Fusion may also be effected by inserting the A.beta.1-6
peptide sequences into a variant of a VLP subunit where part of the
subunit sequence has been deleted, that are further referred to as
truncation mutants. Truncation mutants may have N- or c-terminal,
or internal deletions of part of the sequence of the VLP subunit.
For example, the specific VLP HBcAg with, for example, deletion of
amino acid residues 79 to 81 is a truncation mutant with an
internal deletion. Fusion of A.beta.1-6 peptides to either the N-
or C-terminus of the truncation mutants VLP-subunits also lead to
embodiments of the invention. Likewise, fusion of an epitope into
the sequence of the VLP subunit may also be effected by
substitution, where for example for the specific VLP HBcAg, amino
acids 79-81 are replaced with a foreign epitope. Thus, fusion, as
referred to hereinafter, may be effected by insertion of the
A.beta.1-6 peptide sequence in the sequence of a VLP subunit, by
substitution of part of the sequence of the VLP subunit with the
A.beta.1-6 peptide, or by a combination of deletion, substitution
or insertions.
[0225] The chimeric A.beta.1-6 peptide-VLP subunit will be in
general capable of self-assembly into a VLP. VLP displaying
epitopes fused to their subunits are also herein referred to as
chimeric VLPs. As indicated, the virus-like particle comprises or
alternatively is composed of at least one VLP subunit. In a further
embodiment of the invention, the virus-like particle comprises or
alternatively is composed of a mixture of chimeric VLP subunits and
non-chimeric VLP subunits, i.e. VLP subunits not having an antigen
fused thereto, leading to so called mosaic particles. This may be
advantageous to ensure formation of, and assembly to a VLP. In
those embodiments, the proportion of chimeric VLP-subunits may be
1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95% or higher.
[0226] Flanking amino acid residues may be added to either end of
the sequence of the peptide or epitope to be fused to either end of
the sequence of the subunit of a VLP, or for internal insertion of
such peptidic sequence into the sequence of the subunit of a VLP.
Glycine and serine residues are particularly favored amino acids to
be used in the flanking sequences added to the peptide to be fused.
Glycine residues confer additional flexibility, which may diminish
the potentially destabilizing effect of fusing a foreign sequence
into the sequence of a VLP subunit.
[0227] In a specific embodiment of the invention, the VLP is a
Hepatitis B core antigen VLP. Fusion proteins of the A.beta.1-6
peptide to either the N-terminus of a HBcAg (Neyrinick, S. et al.,
Nature Med 5: 1157-1163 (1999)) or insertions in the so called
major immunodominant region (MIR) have been described (Pumpens, P.
and Grens, E., Intervirology 44: 98-114 (2001)), WO 01/98333), and
are preferred embodiments of the invention. Naturally occurring
variants of HBcAg with deletions in the MIR have also been
described (Pumpens, P. and Grens, E., Intervirology 44: 98-114
(2001), which is expressly incorporated by reference in its
entirety), and fusions to the N- or C-terminus, as well as
insertions at the position of the MIR corresponding to the site of
deletion as compared to a wt HBcAg are further embodiments of the
invention. Fusions to the C-terminus have also been described
(Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001)). One
skilled in the art will easily find guidance on how to construct
fusion proteins using classical molecular biology techniques
(Sambrook, J. et al., eds., Molecular Cloning, A Laboratory Manual,
2nd. edition. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989), Ho et al., Gene 77: 51 (1989)). Vectors and
plasmids encoding HBcAg and HBcAg fusion proteins and useful for
the expression of a HBcAg and HBcAg fusion proteins have been
described (Pumpens, P. & Grens, E. Intervirology 44: 98-114
(2001), Neyrinck, S. et al., Nature Med. 5: 1157-1163 (1999)) and
can be used in the practice of the invention. We also describe by
way of example (Example 6) the insertion of an epitope into the MIR
of HBcAg, resulting in a chimeric self-assembling HBcAg. An
important factor for the optimization of the efficiency of
self-assembly and of the display of the epitope to be inserted in
the MIR of HBcAg is the choice of the insertion site, as well as
the number of amino acids to be deleted from the HBcAg sequence
within the MIR (Pumpens, P. and Grens, E. Intervirology 44: 98-114
(2001); EP 0 421 633; U.S. Pat. No. 6,231,864) upon insertion, or
in other words, which amino acids form HBcAg are to be substituted
with the new epitope. For example, substitution of HBcAg amino
acids 76-80, 79-81, 79-80, 75-85 or 80-81 with foreign epitopes has
been described (Pumpens, P. and Grens, E., Intervirology 44: 98-114
(2001); EP0421635; U.S. Pat. No. 6,231,864). HBcAg contains a long
arginine tail (Pumpens, P. and Grens, E., Intervirology 44: 98-114
(2001)) which is dispensable for capsid assembly and capable of
binding nucleic acids (Pumpens, P. and Grens, E., Intervirology 44:
98-114 (2001)). HBcAg either comprising or lacking this arginine
fall are both embodiments of the invention.
[0228] In a further preferred embodiment of the invention, the VLP
is a VLP of a RNA phage. The major coat proteins of RNA phages
spontaneously assemble into VLPs upon expression in bacteria, and
in particular in E. coli. Specific examples of bacteriophage coat
proteins which can be used to prepare compositions of the invention
include the coat proteins of RNA bacteriophages such as
bacteriophage Q.beta. (SEQ ID NO:4; PIR Database, Accession No.
VCBPQ.beta. referring to Q.beta. CP and SEQ ID NO: 5; Accession No.
AAA16663 referring to Q.beta. A1 protein) and bacteriophage fr (SEQ
ID NO: 7; PIR Accession No. VCBPFR).
[0229] In a more preferred embodiment, the at least one A.beta.1-6
peptide is fused to a Q.beta. coat protein. Fusion protein
constructs wherein epitopes have been fused to the C-terminus of a
truncated form of the A1 protein of Q.beta., or inserted within the
A1 protein have been described (Kozlovska, T. M., et al.,
Intervirology, 39: 9-15 (1996)). The A1 protein is generated by
suppression at the UGA stop codon and has a length of 329 aa, or
328 aa, if the cleavage of the N-terminal methionine is taken into
account. Cleavage of the N-terminal methionine before an alanine
(the second amino acid encoded by the Q.beta. CP gene) usually
takes place in E. coli, and such is the case for N-termini of the
Q.beta. coat proteins. The part of the A1 gene, 3' of the UGA amber
codon encodes the CP extension, which has a length of 195 amino
acids. Insertion of the at least one A.beta.1-6 peptide between
position 72 and 73 of the CP extension leads to further embodiments
of the invention (Kozlovska, T. M., et. al., Intervirology 39: 9-15
(1996)). Fusion of a A.beta.1 -6 peptide at the C-terminus of a
C-terminally truncated Q.beta. A1 protein leads to further
preferred embodiments of the invention. For example, Kozlovska et
al., (Intervirology, 39: 9-15 (1996)) describe Q.beta. A1 protein
fusions where the epitope is fused at the C-terminus of the Q.beta.
CP extension truncated at position 19.
[0230] As described by Kozlovska et al. (Intervirology, 39: 9-15
(1996)), assembly of the particles displaying the fused epitopes
typically requires the presence of both the A1 protein-A.beta.1-6
peptide fusion and the wt CP to form a mosaic particle. However,
embodiments comprising virus-like particles, and hereby in
particular the VLPs of the RNA phage Q.beta. coat protein, which
are exclusively composed of VLP subunits having at least one
A.beta.1-6 peptide fused thereto, are also within the scope of the
present invention.
[0231] The production of mosaic particles may be effected in a
number of ways. Kozlovska et al., Intervirology, 39: 9-15 (1996),
describe three methods, which all can be used in the practice of
the invention. In the first approach, efficient display of the
fused epitope on the VLPs is mediated by the expression of the
plasmid encoding the Q.beta. A1 protein fusion having a UGA stop
codong between CP and CP extension in a E. coli strain harboring a
plasmid encoding a cloned UGA suppressor tRNA which leads to
translation of the UGA codon into Trp (pISM3001 plasmid (Smiley B.
K., et al., Gene 134: 33-40 (1993))). In another approach, the CP
gene stop codon is modified into UAA, and a second plasmid
expressing the A1 protein-A.beta.1-6 peptide fusion is
cotransformed. The second plasmid encodes a different antibiotic
resistance and the origin of replication is compatible with the
first plasmid (Kozlovska, T. M., et al., Intervirology 39: 9-15
(1996)). In a third approach, CP and the A1 protein-A.beta.1-6
peptide fusion are encoded in a bicistronic manner, operatively
linked to a promoter such as the Trp promoter, as described in FIG.
1 of Kozlovska et al., Intervirology, 39: 9-15 (1996).
[0232] In a further embodiment, the A.beta.1-6 peptide is inserted
between amino acid 2 and 3 (numbering of the cleaved CP, that is
wherein the N-terminal methionine is cleaved) of the fr CP, thus
leading to a A.beta.1-6 peptide-fr CP fusion protein. Vectors and
expression systems for construction and expression of fr CP fusion
proteins self-assembling to VLP and useful in the practice of the
invention have been described (Pushko P. et al., Prot. Eng. 6:
883-891 (1993)). In a specific embodiment, the A.beta.1-6 peptide
sequence is inserted into a deletion variant of the fr CP after
amino acid 2, wherein residues 3 and 4 of the fr CP have been
deleted (Pushko P. et al., Prot. Eng. 6: 883-891 (1993)).
[0233] Fusion of epitopes in the N-terminal protuberant
.beta.-hairpin of the coat protein of RNA phage MS-2 and subsequent
presentation of the fused epitope on the self-assembled VLP of RNA
phage MS-2 has also been described (WO 92/13081), and fusion of
A.beta.1-6 peptide by insertion or substitution into the coat
protein of MS-2 RNA phage is also failing under the scope of the
invention.
[0234] In another embodiment of the invention, the A.beta.1-6
peptide is fused to a capsid protein of papillomavirus. To a more
specific embodiment, the A.beta.1-6 peptide is fused to the major
capsid protein L1 of bovine papillomavirus type 1 (BPV-1). Vectors
and expression systems for construction and expression of BPV-1
fusion proteins in a baculovirus/insect cells systems have been
described (Chackerian, B, et al., Proc. Natl. Acad. Sci. USA 96:
2373-2378 (1999), WO 00/23955). Substitution of amino acids 130-136
of BPV-1 L1 with a A.beta.1-6 peptide leads to a BPV-1
L1-A.beta.1-6 peptide, fusion protein, which is a preferred
embodiment of the invention. Cloning in a baculovirus vector and
expression in baculovirus infected Sf9 cells has been described,
and can be used in the practice of the invention (Chackerian, B. et
al., Proc. Natl. Acad. Sci. USA 96: 2373-2378 (1999), WO 00723955).
Purification of the assembled particles displaying the fused
A.beta.1-6 peptide can be performed in a number of ways, such as
for example gel filtration or sucrose gradient ultracentrifugation
(Chackerian, B. et al., Proc. Natl. Acad. Sci. USA 96: 2373-2378
(1999), WO 00/23955).
[0235] In a further embodiment of the invention, the A.beta.1-6
peptide is fused to a Ty protein capable of being incorporated into
a Ty VLP. In a more specific embodiment, the A.beta.1-6 peptide is
fused to the p1 or capsid protein, encoded by the TYA gene (Roth,
J. F., Yeast 16: 785-795 (2000)). The yeast retrotransposons Ty1,
2, 3 and 4 have been isolated from Saccharamyces Serevisiae, while
the retrotransposon Tf1 has been isolated from Schizosaccharomyces
Pombae (Boeke, J. D. and Sandmeyer, S. B., "Yeast Transposable
elements," in The molecular and Cellular Biology of the Yeast
Saccharomyces: Genome dynamics, Protein Synthesis, and Energetics,
p. 193, Cold Spring Harbor Laboratory Press (1991)). The
retrotransposons Ty1 and 2 are related to the copia class of plant
and animal elements, while Ty3 belongs to the gypsy family of
retrotransposons, which is related to plants and animal
retroviruses. In the Ty1 retrotransposon, the p1 protein, also
referred to as Gag or capsid protein, has a length of 440 amino
acids. P1 is cleaved during maturation of the VLP at position 408,
leading to the p2 protein, the essential component of the VLP.
[0236] Fusion proteins to p1 and vectors for the expression of said
fusion proteins in Yeast have been described (Adams, S. E., et al.,
Nature 329: 68-70 (1987)). So, for example, a A.beta.1-6 peptide
may be fused to p1 by inserting a sequence coding for the
A.beta.1-6 peptide into the BamH1 site of the pMA5620 plasmid
(Adams, S. E., et al., Nature 329: 68-70 (1987)). The cloning of
sequences coding for foreign epitopes into the pMA5620 vector leads
to expression of fusion proteins comprising amino acids 1-381 of p1
of Ty1-15, fused C-terminally to the N-terminus of the foreign
epitope. Likewise, N-terminal fusion of a A.beta.1-6 peptide, or
internal insertion into the p1 sequence, or substitution of part of
the p1 sequence are also meant to fall within the scope of the
invention. In particular, insertion of a A.beta.1-6 peptide into
the Ty sequence between amino acids 30-31, 67-68, 113-114 and
132-133 of the Ty protein p1 (EP0677111) leads to preferred
embodiments of the invention.
[0237] Further VLPs suitable for fission of A.beta.1-6 peptides
are, for example, Retrovirus-like-particles (WO9630523), HIV2 Gag
(Rang, Y. C., et al, Biol. Chem. 380: 353-364 (1999)), Cowpea
Mosaic Virus (Taylor, K. M. et al., Biol. Chem. 380: 398-392
(1999)), parvovirus VP2 VLP (Rueda, P. et al., Virology 263: 89-99
(1999)), HBsAg (U.S. Pat. No. 4,722,840, EP0020416B1).
[0238] Examples of chimeric VLPs suitable for the practice of the
invention are also those described in Intervirology 39: 1 (1996).
Further examples of VLPs contemplated for use in the invention are:
HPV-1, HPV-6, HPV-11, HPV-16, HPV-18, HPV-33, HPV-45, CRFV, COPV,
HIV GAG, Tobacco Mosaic Virus. Virus-like particles of SV-40,
Polyomavirus, Adenovirus, Herpes Simplex Virus, Rotavirus and
Norwalk virus have also been made, and chimeric VLPs of those VLPs
comprising a A.beta.1-6 peptide are also within the scope of the
present invention.
[0239] In preferred embodiments of the invention, A.beta.1-6
peptides suitable for generating vaccines of the invention are
modified with an amino acid linker for binding to a VLP. Those
A.beta.1-6 peptides include, but are not limited to: A.beta.1-6
fused C-terminally to the linker GGC. Amino acid linkers suitable
for fusion to the N-terminus of A.beta.1-6 fragments include but
are not limited to the sequence CGG and CGHGNKS. Linkers suitable
for fusion to the C-terminus of A.beta.1-6 include but are not
limited to the sequence GGC. In a preferred embodiment, when a
linker is fused to the C-terminus of A.beta. or A.beta. fragments,
the C-terminal Cysteine is amidated. In a preferred embodiment,
A.beta.1-6 is fused to an amino acid linker and has the sequence:
"NH2-DAEFRHGGC-CONH2, wherein the C-terminal Cysteine is amidated,
which is indicated by the C-terminal "--CONH2", and the N-terminus
of the peptide is free, which is further indicated by "NH2--".
Amino acid linkers are preferably short, to avoid induction of
immune responses against amino acids of said linker, but should
allow the induction of antibodies cross-reactive with soluble
A.beta. and AD plaques and may facilitate the interaction of
antibodies with the A.beta.1-6 peptide. Other suitable properties
of the amino acid linker are flexibility, and preferably lack of
bulky amino acids which might interfere with coupling, and/or
generate an immune response against the linker itself. In more
preferred embodiments, the amino acid linker containing a cysteine
residue as second attachment site is fused to the C-terminus of the
A.beta.1-6 peptide.
[0240] Additional A.beta. fragments suitable in the practice of the
invention include A.beta. fragments corresponding to the
aforementioned fragments, also modified as described above, from
other animal species and eliciting Antibodies cross-reactive with
human amyloid plaques and soluble human A.beta.. Examples of such
fragments are A.beta.1-6 from primates (DAEFRH; SEQ ID NO: 84),
rabbit (DAEFRH: SEQ ID NO: 85), guinea pig (DAEFRH: SEQ ID NO: 88),
mouse (DAEFGH; SEQ ID NO: 76), rat (DAEFGH SEQ ID NO: 87), and
xaenopus laevis (DSEYRH; 86).
[0241] A number of animal models of AD based on transgenic mice
overexpresslng mutated forms of human APP have been reported
(Games, D. et al., Nature 373: 523-527 (1995a): Sturchler-Pierrat
et al., Proc. Natl. Acad. Sci. USA 94: 13287-13292 (1997); Hsiao,
K., et. al., Science 274: 99-102 (1996): Chen, G. et al., Nature
408: 975-979 (2000); Janus, C. et al., Nature 408: 979-982 (2000);
Morgan, D. et al., Nature 408: 982-985 (2000)). Those mice models
differ from each other in the level of overexpression of the
transgene, the AD mutations present on the transgene and the
promoter under which overexpression of the transgene is directed.
These animal models fail to display all of the pathological signs
of AD, which are in particular age-related changes in behaviour,
deposition of .beta.-amyloid into insoluble plaques,
neurofibrillary tangles within neurons, and loss of neurons
throughout the forebrain (Chapman, P. F. Nature 408: 915-916
(2000)). Memory deficits and methods to identify them could however
be identified in those models, and may be used in testing the
effect of the compositions of the invention in animal models (Chen,
G. et al., Nature 408: 975-979 (2000); Janus, C. et al., Nature
408: 979-982 (2000); Morgan, D. et al., Nature 408: 982-985
(2000)). Furthermore, age related deposition of A.beta. into
amyloid plaques can be studied in those models, which also develop
astrocytosis and microgliosis.
[0242] It will be understood by one of ordinary skill in the
relevant arts that other suitable modifications and adaptations to
the methods and applications described herein are readily apparent
and may be trade without departing from the scope of the invention
or any embodiment thereof. Having now described the present
invention in detail, the same will be more clearly understood by
reference to the following examples, which are included herewith
for purposes of illustration only and are not intended to be
limiting of the invention.
EXAMPLES
Example 1
Cloning and Construction, Respectively, Expression and Purification
of Preferred Core Particles and VLP of RNA Phages, Respectively
[0243] A. Construction and Expression of Mutant Q.beta. Coat
Proteins, and Purification of Mutant Q.beta. Coat Protein VLPs or
Capsids.
Plasmid Construction and Cloning of Mutant Coat Proteins
[0244] Construction of pQ.beta.-240:
[0245] The plasmid pQ.beta.10 (Kozlovska, T M, et al., Gene 137:
133-137) was used as an initial plasmid for the construction of
pQ.beta.-240. The mutation Lys13.fwdarw.Arg was created by inverse
PCR. The inverse primers were designed in inverted tail-to-tail
directions:
TABLE-US-00001 (SEQ ID NO: 48) 5'-
GGTAACATCGGTCGAGATGGAAAACAAACTCTGGTCC-3' and (SEQ ID NO: 49) 5'-
GGACCAGAGTTTGTTTTCCATCTCGACCGATGTTACC-3'.
[0246] The products of the first PCR were used as templates for the
second PCR reaction, in which an upstream primer
TABLE-US-00002 5'-AGCTCGCCCGGGGATCCTCTAG-3' (SEQ ID NO: 50)
[0247] and a downstream primer
TABLE-US-00003 (SEQ ID NO: 51) 5'-
CGATGCATTTCATCCTTAGTTATCAATACGCTGGGTTCAG-3'
[0248] were used. The product of the second PCR was digested with
XbaI and MpH1103I and cloned into the pQ.beta.10 expression vector,
which was cleaved by the same restriction enzymes. The PCR
reactions were performed with PCR kit reagents and according to
producer protocol (MBI Fermentas, Vilnius, Lithuania).
[0249] Sequencing using the direct label incorporation method
verified the desired mutations. E. coli cells harbouring
pQ.beta.-240 supported efficient synthesis of 14-kD protein co
migrating upon SDS-PAGE with control Q.beta. coat protein isolated
from Q.beta. phage particles.
TABLE-US-00004 Resulting amino acid sequence: (SEQ ID NO: 17)
AKLETVTLGNIGRDGKQTLVLNPRGVNPTNGVASLSQAGA VP
ALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPS VTRQ
KYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLN PAY
[0250] Construction of pQ.beta.-243:
[0251] The plasmid pQ.beta.10 was used as an initial plasmid for
the construction of pQ.beta.-243. The mutation Asn10.fwdarw.Lys was
created by inverse PCR. The inverse primers were designed in
inverted tail-to-tail directions:
TABLE-US-00005 (SEQ ID NO: 52) 5'-
GGCAAAATTAGAGACTGTTACTTTAGGTAAGATCGG -3' and (SEQ ID NO: 53)
5'-CCGATCTTACCTAAAGTAACAGTCTCTAATTTTGCC- 3'.
[0252] The products of the first PCR were used as templates for the
second PCR reaction, in which an upstream primer
TABLE-US-00006 5'-AGCTCGCCCGGGGATCCTCTAG-3' (SEQ ID NO: 50)
[0253] and a downstream primer
TABLE-US-00007 (SEQ ID NO: 51) 5'-
CGATGCATTTCATCCTTAGTTATCAATACGCTGGGTTCAG-3'
[0254] were used. The product of the second PCR was digested with
XbaI and MpH1103I and cloned into the pQ.beta.10 expression vector,
which was cleaved by the same restriction enzymes. The PCR
reactions were performed with PCR kit reagents and according to
producer protocol (MBI Fermentas, Vilnius, Lithuania).
[0255] Sequencing using the direct label incorporation method
verified the desired mutations. E. coli cells harbouring
pQ.beta.-243 supported efficient synthesis of 14-kD protein co
migrating upon SDSD-PAGE with control Q.beta. coat protein isolated
from Q.beta. phage particles,
TABLE-US-00008 Resulting amino acid sequence: (SEQ ID NO: 18)
AKLETVTLGKIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVP
ALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTQ
KYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY
[0256] Construction of pQ.beta.-250:
[0257] The plasmid pQ.beta.-240 was used as an initial plasmid for
the construction of pQ.beta.-250. The mutation Lys2.fwdarw.Arg was
created by site-directed mutagenesis. An upstream primer
TABLE-US-00009 (SEQ ID NO: 54)
5'-GGCCATGGCACGACTCGAGACTGTTACTTTAGG-3'
[0258] and a downstream primer
TABLE-US-00010 5'-GATTTAGGTGACACTATAG-3' (SEQ ID NO: 55)
[0259] were used for the synthesis of the mutant PCR-fragment,
which was introduced into the pQ.beta.-185 expression vector at the
unique restriction sites NcoI and HindIII. The PCR reactions were
performed with PCR kit reagents and according to producer protocol
(MBI Fermentas, Vilnius, Lithuania).
[0260] Sequencing using the direct label incorporation method
verified the desired mutations. E. coli cells harbouring
pQ.beta.-250 supported efficient synthesis of 14-kD protein co
migrating upon PAGE with control Q.beta. coat protein isolated from
Q.beta. phage particles.
TABLE-US-00011 Resulting amino acid sequence: (SEQ ID NO: 19)
ARLETVTLGNIGRDGKQTLVLNPRGVNPTNGVASLSQAGAVP
ALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQ
KYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY
[0261] Construction of pQ.beta.-251:
[0262] The plasmid pQ.beta.10 was used as an initial plasmid for
the construction of pQ.beta.-251. The mutation Lys16.fwdarw.Arg was
created by inverse PCR. The inverse primers were designed in
inverted tail-to-tail directions:
TABLE-US-00012 (SEQ ID NO: 56) 5'-
GATGGACGTCAAACTCTGGTCCTCAATCCGCGTGGGG -3' and (SEQ ID NO: 57) 5'-
CCCCACGGCGATTGAGGACCAGAGTTTGACGTCCATC -3'.
[0263] The products of the first PCR were used as templates for the
second PCR reaction, in which an upstream primer
TABLE-US-00013 5'-AGCTCGCCCGGGGATCCTCTAG-3' (SEQ ID NO: 50)
[0264] and a downstream primer
TABLE-US-00014 (SEQ ID NO: 51) 5'-
CGATGCATTTCATCCTTAGTTATCAATACGCTGGGTTCAG-3'
[0265] were used. The product of the second PCR was digested with
XbaI and Mph1103I and cloned into the pQ.beta.10 expression vector,
which was cleaved by the same restriction enzymes. The PCR
reactions were performed with PCR kit reagents and according to
producer protocol (MBl Fermentas, Vilnius, Lithuania).
[0266] Sequencing using the direct label incorporation method
verified the desired mutations. E. coli cells harbouring
pQ.beta.-251 supported efficient synthesis of 14-kD protein co
migrating upon SDS-PAGE with control Q.beta. coat protein isolated
from Q.beta. phage particles. The resulting amino acid sequence
encoded by this construct is shown in (SEQ. ID NO: 20).
[0267] Construction of pQ.beta.-259:
[0268] The plasmid pQ.beta.-251 was used as an initial plasmid for
the construction of pQ.beta.-259. The mutation Lys2.fwdarw.Arg was
created by site-directed mutagenesis. An upstream primer
TABLE-US-00015 (SEQ ID NO: 54)
5'-GGCCATGGCACGACTCGAGACTGTTACTTTAGG-3'
and a downstream primer
TABLE-US-00016 5'-GATTTAGGTGACACTATAG-3' (SEQ ID NO: 55)
were used for the synthesis of the mutant PCR-fragment, which was
introduced into the pQ.beta.-185 expression vector at the unique
restriction sites NcoI and HindIII. The PCR reactions were
performed with PCR kit reagents and according to producer protocol
(MBI Fermentes, Vilnius, Lithuania).
[0269] Sequencing using the direct label incorporation method
verified the desired mutations. E. coli cells harbouring
pQ.beta.-259 supported efficient synthesis of 14-kD protein co
migrating upon SDS-PAGE with control Q.beta. coat protein isolated
from Q.beta. phage particles.
TABLE-US-00017 Resulting amino acid sequence: (SEQ ID NO: 21)
AKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVP
ALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQ
KYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY
General Procedures for Expression and Purification of Q.beta. and
Q.beta. Mutants
[0270] Expression
[0271] E. coli JM109 was transformed with Q.beta. coat protein
expression plasmids. 5 ml of LB liquid medium containing 20
.mu.g/ml ampicillin were inoculated with clones transformed with
with Q.beta. coat protein expression plasmids. The inoculated
culture was incubated at 3.degree. C. for 16-24 h without shaking.
The prepared inoculum was subsequently diluted 1:100 in 100-300 ml
of fresh LB medium, containing 20 .mu.g/ml ampicillin, and
incubated at 37.degree. C. overnight without shaking. The resulting
second inoculum was diluted 1:50 in M9 medium containing 1%
Casamino acids and 0.2% glucose in flasks, and incubated at
37.degree. C. overnight under shaking.
Purification
[0272] Solutions and buffers for the purification procedure; 1.
Lysis buffer LB 50 mM Tris-HCl pH8.0 with 5 mM EDTA, 0.1% [0273]
triton X100 and freshly prepared PMSF at a concentration of 5
micrograms per ml. Without lysozyme and DNAse.
2. SAS
[0273] [0274] Saturated ammonium sulphate in water
3. Buffer NET.
[0274] [0275] 20 mM Tris-HCl, pH 7.8 with 5 mM EDTA and 150 mM
NaCl.
4. PEG
[0275] [0276] 40% (w/v) polyethylenglycol 6000 in NET
Disruption and Lysis
[0277] Frozen cells were resuspended in LB at 2 ml/g cells. The
mixture was sonicated with 22 kH five times for 15 seconds, with
intervals of 1 min to cool the solution on ice. The lysate was then
centrifuged at 14 000 rpm, for 1h using a Janecki K 60 rotor. The
centrifugation steps described below were all performed using the
same rotor, except otherwise stated. The supernatant was stored at
4.degree. C., while cell debris were washed twice with LB. After
centrifugation, the supernatants of the lysate and wash fractions
were pooled.
[0278] Fractionation
[0279] A saturated ammonium sulphate solution was added dropwise
under stirring to the above pooled lysate. The volume of the SAS
was adjusted to be one fifth of total volume, to obtain 20% of
saturation. The solution was left standing overnight, and was
centrifuged the next day at 14 000 rpm, for 20 min. The pellet was
washed with a small amount of 20% ammonium sulphate, and
centrifuged again. The obtained supernatants were pooled, and SAS
was added dropwise to obtain 40% of saturation. The solution was
left standing overnight and was centrifuged the next day at 14 000
rpm, for 20 min. The obtained pellet was solubilised in NET
buffer.
[0280] Chromatography
[0281] The capsid or VLP protein resolubilized in NET buffer was
loaded on a Sepharose. CL-4B column. Three peaks eluted during
chromatography. The first one mainly contained membranes and
membrane fragments, and was not collected. Capsids were contained
in the second peak, while the third one contained other E. coli
proteins.
[0282] The peak fractions were pooled, and the NaCl concentration
was adjusted to a final concentration of 0.65 M. A volume of PEG
solution corresponding to one half of the pooled peak fraction was
added dropwise under stirring. The solution was left to stand
overnight without stirring. The capsid protein was sedimented by
centrifugation at 14 000 rpm for 20 min. It was then solubilized in
a minimal volume of NET and loaded again on the Sepharose CL-4B
column. The peak fractions were pooled, and precipitated with
ammonium sulphate at 60% of saturation (w/v). After centrifugation
and resolubilization in NET buffer, capsid protein was loaded on a
Sepharose CL-6B column for rechromatography.
[0283] Dialysis and Drying
[0284] The peak fractions obtained above were pooled and
extensively dialysed against sterile water, and lyophilized for
storage.
[0285] Expression and Purification Q.beta.-240
[0286] Cells (E. coli JM 109, transformed with the plasmid
pQ.beta.-240) were resuspended in LB, sonicated five times for 15
seconds (water ice jacket) and centrifuged at 13000 rpm for one
hour. The supernatant was stored at 4.degree. C. until further
processing, while the debris were washed 2 times with 9 ml of LB,
and finally with 9 ml of 0.7 M urea in LB. All supernatants were
pooled, and loaded on the Sepharose CL-4B column. The pooled peak
fractions were precipitated with ammonium sulphate and centrifuged.
The resolubilized protein was then purified further on a Sepharose
2B column and finally on a Sepharose 6B column. The capsid peak was
finally extensively dialyzed against water and lyophilized as
described above. The assembly of the coat protein into a capsid was
confirmed by electron microscopy.
[0287] Expression and Purification Q.beta.-243
[0288] Cells (E. coli RRI) were resuspended in LB and processed as
described in the general procedure., The protein was purified by
two successive gel filtration steps on the sepharose CL-4B column
and finally on a sepharose CL-2B column. Peak fractions were pooled
and lyophilized as described above. The assembly of the coat
protein into a capsid was confirmed by electron microscopy.
[0289] Expression and Purification of Q.beta.-250
[0290] Cells (E. coli JM 109, transformed with pQ.beta.-250) were
resuspended in LB and processed as described above. The protein was
purified by gel filtration on a Sepharose CL-4B and finally on a
Sepharose CL-2B column, and lyophilized as described above. The
assembly of the coat protein into a capsid was confirmed by
electron microscopy.
[0291] Expression and Purification of Q.beta.-259
[0292] Cells (E. coli JM 109, transformed with pQ.beta.-259 ) were
re-suspended in LB and sonicated. The debris were washed once with
10 ml of LB and a second time with 10 ml of 0.7 M urea in LB. The
protein was purified by two gel-filtration chromatography steps, on
a Sepharose CL-4 B column. The protein was dialyzed and
lyophilized, as described above. The assembly of the coat protein
into a capsid was confirmed by electron microscopy.
B. Cloning, Expression and Purification of Recombinant AP205
VLP
Cloning of the AP205 Coat Protein Gene
[0293] The cDNA of AP205 coat protein (CP) (SEQ ID NO: 28) was
assembled from two cDNA fragments generated from phage AP205 RNA by
using a reverse transcription-PCR technique and cloning in the
commercial plasmid pCR 4-TOPO for sequencing. Reverse transcription
techniques are well known to those of ordinary skill in the
relevant art. The first fragment, contained in plasmid p205-246,
contained 269 nucleotides upstream of the CP sequence and 74
nucleotides coding for the first 24 N-terminal amino acids of the
CP. The second fragment, contained in plasmid p205-262, contained
364 nucleotides coding for amino acids 12-131 of CP and an
additional 162 nucleotides downstream of the CP sequence. Both
p205-246 and p205-262 were a generous gift from J. Klovins.
[0294] The plasmid 283.-58 was designed by two-step PCR, in order
to fuse both CP fragments from plasmids p205-246 and p205-262 in
one full-length CP sequence.
[0295] An upstream primer p1.44 containing the NcoI site for
cloning into plasmid pQb185, or p1.45 containing the XbaI site for
cloning into plasmid pQb10, and a downstream primer p1.46
containing the HindIII restriction site were used (recognition
sequence of the restriction enzyme underlined):
TABLE-US-00018 p1.44 (SEQ ID NO: 79) 5'-AACC ATG GCA AAT AAG CCA
ATG CAA CCG-3' P1.45 (SEQ ID NO: 80)
5'-AATCTAGAATTTTCTGCGCACCCATCCCGG-3' P1.46 (SEQ ID NO: 81) 5'-AAAAG
TTA AGC AGT AGT ATC AGA CGA TAC G-3'
[0296] Two additional primers, p1.47, annealing at the 5' end of
the fragment contained in p205-262, and p1.48, annealing at the 3'
end of the fragment contained in plasmid p205-246 were used to
amplify the fragments in the first PCR. Primers p1.47 and p1.48 are
complementary to each other.
TABLE-US-00019 p1.47: (SEQ ID NO: 82)
5'-GAGTGATCCAACTCGTTTATCAACTACATTT- TCAGCAAGTCTG-3' p1.48: (SEQ ID
NO: 83) 5'-CAGACTTGCTGAAAATGTAGTTGATAAACGA- GTTGGATCACTC-3'
[0297] In the first two PCR reactions, two fragments were
generated. The first fragment was generated with primers p1.45 and
p1.48 and template p205-246. The second fragment was generated with
primers p1.47 and p1.46, and template p205-262. Both fragments were
used as templates for the second PCR reaction, a splice-overlap
extension, with the primer combination p1.45 and p1.46 or p1.44 and
p1.46 . The product of the two second-step PCR reactions were
digested with XbaI or NcoI respectively, and HindIII and cloned
with the same restriction sites into pQb10 or pQb185 respectively,
two pGEM-derived expression, vectors under the control of E. coli
tryptophan operon promoter.
[0298] Two plasmids were obtained, pAP283-58 (SEQ ID NO: 27),
containing the gene coding for wt AP205 CP (SEQ ID NO: 28) in
pQb10, and pAP281-32 (SEQ ID NO: 30) with mutation Pro5.fwdarw.Thr
(SEQ ID NO: 29), in pQb185. The coat protein sequences were
verified by DNA sequencing. PAP283-58 contains 49 nucleotides
upstream of the ATG codon of the CP, downstream of the XbaI site,
and contains the putative original ribosomal binding site of the
coat protein mRNA.
Expression and Purification of Recombinant AP205 VLP
[0299] A. Expression of Recombinant AP205 VLP
[0300] E. coli JM109 was transformed with plasmid pAP283-58. 5 ml
of LB liquid medium with 20 .mu.g/ml ampicillin were inoculated
with a single colony, and incubated at 37.degree. C. for 16-24 h
without shaking.
[0301] The prepared inoculum was diluted 1:100 in 100-300 ml of LB
medium, containing 20 .mu.g/ml ampicillin and incubated at
37.degree. C. overnight without shaking. The resulting second
inoculum was diluted 1:50 in 2TY medium, containing 0.2% glucose
and phosphate for buffering, and incubated at 37.degree. C.
overnight on a shaker. Cells were harvested by centrifugation and
frozen at -80.degree. C.
[0302] B. Purification of Recombinant AP205 VLP [0303] Solutions
and buffers:
[0304] 1. Lysis buffer
[0305] 50 mM Tris-HCl pH 8.0 with 5 mM EDTA, 0.1% tritonX100 and
PMSF at 5 micrograms per ml
[0306] 2. SAS [0307] Saturated ammonium sulphate in water
[0308] 3. Buffer NET. [0309] 20 mM Tris-HCl, pH 7.8 with 5 mM EDTA
and 150 mM NaCl.
[0310] 4. PEG [0311] 40% (w/v) polyethylenglycol 6000 in NET
Lysis:
[0312] Frozen cells were resuspended in lysis buffer at 2 ml/g
cells. The mixture was sonicated with 22 kH five times for 15
seconds, with intervals of 1 min to cool the solution on ice. The
lysate was then centrifuged for 20 minutes at 12 000 rpm, using a
F34-6-38 rotor (Ependorf). The centrifugation steps described below
were all performed using the same rotor, except otherwise stated.
The supernatant was stored at 4.degree. C., while cell debris were
washed twice with lysis buffer. After centrifugation, the
supernatants of the lysate and wash fractions were pooled.
[0313] Ammonium-sulphate precipitation can be further used to
purify AP205 VLP. In a first step, a concentration of
ammonium-sulphate at which AP205 VLP does not precipitate is
chosen. The resulting pellet is discarded. In the next step, an
ammonium sulphate concentration at which AP205 VLP quantitatively
precipitates is selected, and AP205 VLP is isolated from the pellet
of this precipitation step by centrifugation (14 000 rpm, for 20
min). The obtained pellet is solubilized in NET buffer.
Chromatography:
[0314] The capsid protein from the pooled supernatants was loaded
on a Sepharose 4B column (2.8.times.70 cm), and eluted with NET
butter, at 4 ml/hour/fraction. Fractions 28-40 were collected, and
precipitated with ammonium sulphate at 60% saturation. The
fractious were analyzed by SDS-PAGE and Western Blot with an
antiserum specific for AP205 prior to precipitation. The pellet
isolated by centrifugation was resolubilized in NET buffer, and
loaded on a Sepharose 2B column (2.3.times.65 cm), eluted at 3
ml/h/fraction. Fractions were analysed by SDS-PAGE, and fractions
44-50 were collected, pooled and precipitated with ammonium
sulphate at 60% saturation. The pellet isolated by centrifugation
was resolubilized in NET buffer, and purified on a Sepharose 6B
column (2.3.times.47 cm), eluted at 3 ml/hour/fraction. The
fractions were analysed by SDS-PAGE. Fractions 23-27 were
collected, the salt concentration adjusted to 0.5 M, and
precipitated with PEG 6000, added from a 40% stock in water and to
a final concentration of 13.3%. The pellet isolated by
centrifugation was resolubilized in NET buffer, and loaded on the
same Sepharose 2B column as above, eluted in the same manner.
Fractions 43-53 were collected, and precipitated with ammonium
sulphate at a saturation of 60%. The pellet isolated by
centrifugation was resolubilized in water, and the obtained protein
solution was extensively dialyzed against water. About 10 mg of
purified protein per gram of cells could be isolated.
[0315] Examination of the virus-like particles in Electron
microscopy showed that they were identical to the phage
particles.
Example 2
Insertion of a Peptide Containing a Lysine Residue into the c/e1
Epitope of HBcAg(1-149)
[0316] The c/e1 epitope (residues 72 to 88) of HBcAg is located in
the tip region on the surface of the Hepatitis B virus capsid
(HBcAg). A part of this region (Proline 79 and Alanine 80) was
genetically replaced by the peptide Gly-Gly-Lys-Gly-Gly (SEQ ID NO:
33), resulting in the HBcAg-Lys construct (SEQ ID NO: 26). The
introduced Lysine residue contains a reactive amino group in its
side chain that can be used for intermolecular chemical
crosslinking of HBcAg particles with any antigen containing a free
cysteine group.
[0317] HBcAg-Lys DNA, having the amino acid sequence shown in SEQ
ID NO:78, was generated by PCRs: The two fragments encoding HBcAg
fragments (amino acid residues 1to 78 and 81 to 149) were amplified
separately by PCR. The primers used for these PCRs also introduced
a DNA sequence encoding the Gly-Gly-Lys-Gly-Gly peptide (SEQ ID NO:
33). The HBcAg (1 to 78) fragment was amplified from pEco63 using
primers EcoRIHBcAg(s) and Lys-HBcAg(as). The HBcAg (81 to 149)
fragment was amplified from pEco63 using primers Lys-HBcAg(s) and
HBcAg(1-149)Hind(as). Primers Lys-HBcAg(as) and Lys-HBcAg(s)
introduced complementary DNA sequences at the ends of the two PCR
products allowing fusion of the two PCR products in a subsequent
assembly PCR. The assembled fragments were amplified by PCR using
primers EcoRIHBcAg(s) and HbcAg(1-149)Hind(as).
[0318] For the PCRs, 100 pmol of each oligo and 5.0 ng of the
template DNAs were used in the 50 -ml reaction mixtures with 2
units of Pwo polymerase, 0.1 mM dNTPs and 2 mM MgSO4. For both
reactions, temperature cycling was carried out as follows:
94.degree. C. for 2 minutes; 30 cycles of 94.degree. C. (1 minute),
50.degree. C. (1 minute), 72.degree. C. (2 minutes).
[0319] Primer Sequences:
TABLE-US-00020 EcoRIHBcAg(s): (SEQ ID NO: 58)
(5'-CCGGAATTCATGGACATTGACCCTTATAAAG-3'); Lys-HBcAg(as): (SEQ ID NO:
59) (5'-CCTAGAGCCACCTTTGCCACCATCTTCTAAATTAG- TACCCACCCAGGTAGC-3');
Lys-HBcAg(s): (SEQ ID NO: 60) (5'-GAAGATGGTGGCAAAGGTGGCTCTAGGGACC-
TAGTAGTCAGTTATGTC-3'); HBcAg(1-149)Hind(as): (SEQ ID NO: 61)
(5'-CGCGTCCCAAGCTTCTAAACAACAGTAGTCTCCGGAAG-3').
[0320] For fusion, of the two PCR fragments by PCR 100 pmol of
primers EcoRIHBcAg(s) and HBcAg(1-149)Hind(as) were used with 100
ng of the two purified PCR fragments in a 50 ml reaction mixture
containing 2 units of Pwo polymerase, 0.1 mM dNTPs and 2 mM
MgSO.sub.4. PCR cycling conditions were: 94.degree. C. for 2
minutes; 30 cycles of 94.degree. C. (1 minute), 50.degree. C. (1
minute), 72.degree. C. (2 minutes). The assembled PCR product was
analyzed by agarose gel electrophoresis, purified and digested for
19 hours in an appropriate buffer with EcoRI and HindIII
restriction enzymes. The digested DNA fragment was ligated into
EcoRI/HindIII-digested pKK vector to generate pKK-HBcAg-Lys
expression vector. Insertion of the PCR product into the vector was
analyzed by EcoRI/HindIII restriction analysis and DNA sequencing
of the insert.
Example 3
Expression and Purification of HBcAg-Lys
[0321] E. coli strains K802 or JM109 were transformed with
pKK-HBcAg-Lys. 1 ml of an overnight culture of bacteria was used to
innolculate 100 ml of LB medium containing 100 .mu.g/ml ampicillin.
This culture was grown for 4 hours at 37.degree. C. until an OD at
600 nm of approximately 0.8 was reached. Induction of the synthesis
of HBcAg-Lys was performed by addition of IPTG to a final
concentration of 1 mM. After induction, bacteria were further
shaken at 37.degree. C. for 4 hours. Bacteria were harvested by
centrifugation at 5000.times.g for 15 minutes. The pellet was
frozen at -80.degree. C. The pellet was thawed and resuspeaded in
bacteria lysis buffer (10 mM NaHPO.sub.4, pH 7.0, 30 mM NaCl, 0.25%
Tween-20, 10 mM EDTA) supplemented with 200 .mu.g/ml lysozyme and
10 .mu.l of Benzonase (Merck). Cells were incubated for 30 minutes
at room temperature and disrupted by sonication. E. coli cells
harboring pKK-HBcAg-Lys expression plasmid or a control plasmid
were used for induction of HBcAg-Lys expression with IPTG. Prior to
the addition of IPTG, a sample was removed from the bacteria
culture carrying the pKK-HBcAg-Lys plasmid and from a culture
carrying the control plasmid. Four hours after addition of IPTG,
samples were again removed from the culture containing
pKK-HBcAg-Lys and from the control culture. Protein expression was
monitored by SDS-PAGE followed by Coomassie staining.
[0322] The lysate was then centrifuged for 30 minutes at
12,000.times.g in order to remove insoluble cell debris. The
supernatant and the pellet were analyzed by Western blotting using
a monoclonal antibody against HBcAg (YVS1841, purchased from
Accurate Chemical and Scientific Corp., Westbury, N.Y., USA),
indicating that a significant amount of HBcAg-Lys protein was
soluble. Briefly, lysates from E. coli cells expressing HBcAg-Lys
and from control cells were centrifuged at 14,000.times.g for 30
minutes. Supernatant (=soluble fraction) and pellet (=insoluble
fraction) were separated and diluted with SDS sample buffer to
equal volumes. Samples were analyzed by SDS-PAGE followed by
Western blotting with anti-HBcAg monoclonal antibody YVS 1841.
[0323] The cleared cell lysate was used for step-gradient
centrifugation using a sucrose step gradient consisting of a 4 ml
65% sucrose solution overlaid with 3 ml 15% sucrose solution
followed by 4 ml of bacterial lysate. The sample was centrifuged
for 3 hrs with 100,000.times.g at 4.degree. C. After
centrifugation, 1 ml fractious from the top of the gradient were
collected and analyzed by SDS-PAGE followed by Coomassie staining.
The HBcAg-Lys protein was detected by Coomassie staining.
[0324] The HBcAg-Lys protein was enriched at the interface between
15 and 65% sucrose indicating that it had formed a capsid particle.
Most of the bacterial proteins remained in the sucrose-free upper
layer of the gradient, therefore step-gradient centrifugation of
the HBcAg-Lys particles led both to enrichment and to a partial
purification of the particles.
[0325] Expression and purification of HBcAg-Lys in large scale was
performed as follows. An overnight culture was prepared by
inoculating a single colony in 100 ml LB, 100 .mu.g/ml Ampicillin
and growing the culture overnight at 37.degree. C. 25 ml of the
preculture were diluted in 800 ml LB Ampicillin medium the next
day, and the culture grown to an optical density OD.sup.600 of
0.6-0.8. The culture was then induced with 1 mM IPTG, and left to
grow for another 4 hours. The cells were harvested and lysed
essentially as described above.
[0326] HBcAg-Lys was then purified by first precipitating the
protein with ammonium sulphate (30% saturation) from the cleared
cell lysate, then loading the resolubilized pellet on a gel
filtration column (Sephacryl S-400, Pharmacia). The pooled
fractions were precipitated again with ammonium sulphate, the
pellet resolubilized and loaded a second time on the same gel
filtration column. The fractions were finally pooled and
concentrated, and the concentration assessed using a Bradford test
(BioRad).
Example 4
Construction of a HBcAg Devoid of Free Cysteine Residues and
Containing an Inserted Lysine Residue
[0327] A Hepatitis core Antigen (HBcAg), referred to herein as
HBcAg-lys-2cys-Mut, devoid of cysteine residues at positions
corresponding to 48 and 107 in SEQ ID NO:25 and containing an
inserted lysine residue was constructed using the following
methods.
[0328] The two mutations were introduced by first separately
amplifying three fragments of the HBcAg-Lys gene prepared as
described above in Example 2 with the following PCR primer
combinations. PCR methods and conventional cloning techniques were
used to-prepare the HBcAg-lys2cys-Mut gene.
[0329] In brief, the following primers were used to prepare
fragment 1:
TABLE-US-00021 Primer 1: EcoRIHBcAg(s) (SEQ ID NO: 58)
CCGGAATTCATGGACATTGACCCTTATAAAG Primer 2: 48as (SEQ ID NO: 62)
GTGCAGTATGGTGAGGTGAGGAATGCTCAGGAGACTC
[0330] The following primers were used to prepare fragment 2:
TABLE-US-00022 Primer 3: 48s (SEQ ID NO: 63)
GSGTCTCCTGAGCATTCCTCACCTCACCATACTGCAC Primer 4: 107as (SEQ ID NO:
64) CTTCCAAAAGTGAGGGAAGAAATGTGAAACCAC
[0331] The following primers were used to prepare fragment 3:
TABLE-US-00023 Primer 5: HBcAg149hind-as (SEQ ID NO: 65)
CGCGTCCCAAGCTTCTAAACAACAGTAGTGTCCGGAAGC- GTTGATAG Primer 6: 107s
(SEQ ID NO: 66) GTGGTTTCACATTTCTTCCCTCACTTTTGGAAG
[0332] Fragments 1 and 2 were then combined with PCR primers
EcoRIHBcAG(s) and 107 as to give fragment 4. Fragment 4 and
fragment 3 were then combined with primers EcoRIHBcAg(s) and
HBcAgI49hind--as to produce the full length gene. The full length
gene was then digested with the EcoRI (GAATTC) and HindIII (AAGCTT)
enzymes and cloned into the pKK vector (Pharmacia) cut at the same
restriction, sites. Expression and purification of
HBcAg-lys-2cys-Mut were performed as set out in Example 3.
Example 5
Construction of HBcAG1-185-Lys
[0333] Hepatitis core Antigen (HBcAg) 1-185 was modified as
described in Example 2. A part of the c/e1 epitope (residues 72 to
88) region (Proline 79 and Alanine 80) was genetically replaced by
the peptide Gly-Gly-Lys-Gly-Gly (SEQ ID NO: 33), resulting in the
HBcAg-Lys construct (SEQ ID NO: 26). The introduced Lysine residue
contains a reactive amino group in its side chain that can be used
for intermolecular chemical crosslinking of HBcAg particles with
any antigen containing a tree cysteine group. PCR methods and
conventional cloning techniques were used to prepare the
HBcAg1-185-Lys gene.
[0334] The Gly-Gly-Lys-Gly-Gly sequence (SEQ ID NO: 33) was
inserted by amplifying two separate fragments of the HBcAg gene
from pEco63, as described above in Example 2 and subsequently
fusing the two fragments by PCR to assemble the full length gene.
The following PCR primer combinations were used:
TABLE-US-00024 fragment 1: Primer 1: EcoRIHBcAg(s) (SEQ ID NO: 58)
(see Example 2) Primer 2: Lys-HBcAg(as) (SEQ ID NO: 59) (see
Example 2) fragment 2: Primer 3: Lys-HBcAg(s) (SEQ ID NO: 60) (see
Example 2) Primer 4: HBcAgwtHindIIII (SEQ ID NO: 67)
CGCGTCCCAAGCTTCTAACATTGAGATTCCCGAGATTG Assembly: Primer 1:
EcoRIHBcAg(s) (SEQ ID NO: 58) (see example 2) Primer 2:
HBcAgwtHindIIII (SEQ ID NO: 67)
[0335] The assembled full length gene was then digested with the
EcoRI (GAATTC) and HindIII (AAGCTT) enzymes and cloned into the pKK
vector (Pharmacia) cut at the same restriction sites.
Example 6
Fusion of a Peptide Epitope in the MIR Region of HbcAg
[0336] The residues 79 and 80 of HBcAg1-185 were substituted with
the epitope C.epsilon.H3 of sequence VNLTWSRASG (SEQ ID NO: 68).
The C.epsilon.H3 sequence stems from the sequence of the third
constant domain of the heavy chain of human IgE. The epitope was
inserted in the HBcAgI-185 sequence using an assembly PCR method.
In the first PCR step, the HBcAg1-185 gene originating from ATCC
clone pEco63 and amplified with primers HBcAg-wt EcoRI fwd and
HBcAg-wt Hind III rev was used as template in two separate
reactions to amplify two fragments containing sequence elements
coding for the C.epsilon.H3 sequence. These two fragments were then
assembled in a second PCR step, in an assembly PCR reaction.
[0337] Primer combinations in the first PCR step: C.epsilon.H3fwd
with HBcAg-wt Hind III rev, and HBcAg-wt EcoRI fwd with
C.epsilon.H3ref. In the assembly PCR reaction, the two fragments
isolated in the first PCR step were first assembled during 3 PCR
cycles without outer primers, which were added afterwards to the
reaction mixture for the next 25 cycles. Outer primers: HBcAg-wt
EcoRI fwd and HBcAg-wt Hind III rev.
[0338] The PCR product was cloned in the pKK223.3 using the EcoRI
and HindIII sites, for expression in E. coli (see Example 2). The
chimeric VLP was expressed in E. coli and purified as described in
Example 2. The elution volume at which the HBcAG1-185-C.epsilon.H3
eluted from the gel filtration showed assembly of the fusion
proteins to a chimeric VLP.
Primer Sequences:
TABLE-US-00025 [0339] C.epsilon.H3fwd: (SEQ ID NO: 69) 5' GTT AAC
TTG ACC TGG TCT CGT GCT TCT GGT GCA TCC AGG GAT CTA GTA GTC 3' (SEQ
ID NO: 70) V N L T W S R A S G A80 S R D L V V86 C.epsilon.H3rev:
(SEQ ID NO: 71) 5' ACC AGA AGC ACG AGA CCA GGT CAA GTT AAC ATC TTC
CAA ATT ATT ACC CAC 3' (SEQ ID NO: 72) D78 E L N N G V72 HBcAg-wt
EcoRI fwd: (SEQ ID NO: 73) 5' CCGgaattcATGGACATTGACCCTTATAAAG
HBcAg-wt Hind III rev: (SEQ ID NO: 74) 5'
CGCGTCCCaagcttCTAACATTGAGATTCCCGAGATTG
Example 7
Fusion of A.beta.1-6 Peptide in the MIR Region of HbcAg
[0340] The residues 79 and 80 of HBcAg1-185 are substituted with
the A.beta.1-6 peptide of sequence DAEFRH (SEQ ID NO: 75) or DAEFGH
(SEQ ID NO: 76). Two overlapping printers are designed using the
same strategy described in Example 6, and the fusion protein
constructed by assembly PCR. The PCR product is cloned in the
pKK223.3 vector, and expressed in E. coli K802. The chimeric VLPs
are expressed and purified as described in Example 3.
Example 8
Fusion of a A.beta.1-6 Peptide to the C-Terminus of the Q.beta. A1
Protein Truncated at Position 19 of the CP Extension
[0341] A primer annealing to the 5' end of the Q.beta. A1 gene and
a primer annealing to the 3' end of the A1 gene and comprising
additionally a sequence element coding for the A.beta.1-6 peptide,
of sequence DAEFRH (SEQ ID NO: 75) or DAEFGH (SEQ ID NO: 76), are
used in a PCR reaction with pQ.beta.10 as template. The PCR product
is cloned in pQ.beta.10 (Kozlovska T. M. et al., Gene 137: 133-37
(1993)), and the chimeric VLP expressed and purified as described
in Example 1.
Example 9
Insertion of a A.beta.1-6 Peptide Between Positions 2 and 3 of fr
Coat Protein
[0342] Complementary primers coding for the sequence of the
A.beta.1-6 peptide of sequence DAEFRH (SEQ ID NO: 75) or DAEFGH
(SEQ ID NO: 76), and containing Bsp119I compatible ends and
additional nucleotides enabling in frame insertion, are inserted in
the Bsp119I site of the pFrd8 vector (Pushko, P. et al., Prot. Eng.
6: 883-91 (1993)) by standard molecular biology techniques.
Alternatively, the overhangs of the pFrd8 vector are filled in with
Klenow after digestion with Bsp119I, and oligonucleotide coding for
the sequence of the A.beta.1-6 peptide and additional nucleotides
for in frame cloning are ligated in pFrd8 after the Klenow
treatment. Clones with the insert in the right orientation are
analysed by sequencing. Expression and purification of the chimeric
fusion protein in E. coli JM109 or E. coli K802 is performed as
described in Pushko, P. et al. Prot. Eng. 6: 883-91 (1993), but for
the chromatography steps which are performed using a Sepharose
CL-4B or Sephacryl S-400 (Pharmacia) column. The cell lysate is
precipitated with ammonium sulphate, and purified by two successive
gel filtration purification steps, similarly to the procedure
described for Q.beta. in Example 1.
Example 10
Insertion of a A.beta.1-6 Peptide Between Positions 6 and 68 of Ty1
Protein p1 in the Vector pOGS8111
[0343] Two complementary oligonucleotides coding for the A.beta.1-6
peptide, of sequence DAEFRH (SEQ ID NO: 75) or DAEFGH (SEQ ID NO:
76), with ends compatible with the NheI site of pOGS8111 are
synthesized. Additional nucleotides are added to allow for in frame
insertion of a sequence coding for the A.beta.1-6 peptide according
to the description of EP 677,111. The amino acids AS and SS
flanking the inserted epitope are encoded by the altered NheI sites
resulting from the insertion of the oligonucleotide in the TyA(d)
gene of pOGS8111.
[0344] POGS8111 is transformed into S. cervisiae strain MC2, for
expression of the chimeric Ty VLP as described in EP0677111 and
references therein. The chimeric Ty VLP is purified by sucrose
gradient ultracentrifugation as described in EP677,111.
Example 11
Insertion of a A.beta.1-6 Peptide into the Major Capsid Protein L1
of Papillomavirus Type I (BPV-1)
[0345] A sequence coding for the A.beta.1-6 peptide having the
sequence DAEFRH (SEQ ID NO: 75) or DAEFGH (SEQ ID NO: 76) is
substituted to the sequence coding for amino acids 130-136 of the
BPV-1 L1 gene cloned in the pFastBac1 (GIBCO/BRL) vector as
described (Chackerian, B. el al., Proc. Natl. Acad. USA 96:
2373-2378 (1999)). The sequence of the construct is verified by
nucleotide sequence analysis. Recombinant baculovirus is generated
using the GIBCO/BRL baculovirus system, as described by the
manufacturer. The chimeric VLPs are purified, from baculovirus
infected Sf9 cells as described by Kirnbauer, R. et al., Proc.
Natl. Acad. Sci. 89: 12180-84 (1992) and Greenstone, H. L. et al.,
Proc. Natl. Acad. Sci. 95: 1800-05 (1998).
Example 12
Immunization of Mice with A.beta.1-6 Peptide Fused to VLPs
[0346] Chimeric VLPs displaying the A.beta.1-6 peptide of sequence
DAEFRH (SEQ ID NO: 75) or DAEFGH (SEQ ID NO: 76) generated in
Examples 7-11 are used for immunization of human transgenic APP
mice or C57/BL6 mice as described in Example 13 and 14. The sera
obtained from the immunized mice are analysed in a A.beta.1-6
peptide or A.beta.1-40 or A.beta.1-42 specific ELISA as described
in Example 13.
[0347] The protective effect of the vaccine is examined by
immunizing a large group of human APP transgenic mice as described
in Example 14.
Example 13
Coupling of A.beta.1-6 Peptide to Q.beta. VLP (Q.beta.A.beta.1-6),
and Immunization of Mice with Q.beta.A.beta.1-6
A. Coupling of A.beta.1-6 Peptide Q.beta. VLP
[0348] The A.beta.1-6 peptide (sequence: NH2-DAEFRHGGC-CONH2) (SEQ
ID NO: 77) was chemically synthesized; the initial NH2 group
indicates that the peptide has a free N-terminus, and the terminal
NH2 group indicates that the peptide has an amidated
carboxy-terminus. Q.beta. VLP was expressed and purified as
described in example 1. Q.beta. VLP, in 20 mM Hepes, 150 mM NaCl,
pH 8.2 (HBS, pH 8.2) was reacted at a concentration of 2 mg/ml
(determined in a Bradford assay), with 1.43 mM SMPH (Pierce,
Rockford, Ill.), diluted from a stock in DMSO, for 30 minutes at
room temperature (RT). The reaction mixture was then dialyzed
against HBS, pH 8.2 buffer at 4.degree. C., and reacted with 0.36
mM of A.beta.1-6 peptide, diluted in the reaction mixture from a 50
mM stock in DMSO. The coupling reaction was left to proceed for 2
hours at 15.degree. C., and the reaction mixture dialyzed 2.times.2
hours against a 1000-fold volume HBS, pH 8.2, and flash frozen in
liquid nitrogen in aliquots for storage at -80.degree. C. until
further use.
[0349] An aliquot was thawed, and coupling of the A.beta.1-6
peptide to the Q.beta. VLP subunits assessed by SDS-PAGE and the
protein concentration measured in a Bradford assay. The result of
the coupling reactions are shown in FIG. 1.
[0350] FIG. 1 shows the SDS-PAGE analysis of the coupling reaction
of A.beta.1-6 peptide and Q.beta. VLP. The samples were run under
reducing conditions on a 16% Tris-glycine gel, stained with
coomassie brilliant blue. Lane 1 is the protein marker, with
corresponding molecular weights indicated on the left border of the
gel; lane 2, derivatized Q.beta. VLP protein; lane 3, the
supernatant of the coupling reaction of Q.beta. VLP protein to the
A.beta.1-6 peptide; lane 4, the pellet of the coupling reaction of
Q.beta. VLP protein to the ABL6 peptide; Coupling products
corresponding to the coupling of 1, 2 and 3 peptides per monomer
are indicated by arrows in the Figure. More than 1.5-peptides per
subunit were coupled on average; nearly no subunits were left
uncoupled.
B. Immunisation of Mice with A.beta.1-6 peptide coupled to Q.beta.
VLP and Analysis of Immune Response
[0351] Q.beta. VLP coupled to A.beta.1-6 peptide (denominated here
Qb-Ab-1-6) was injected s.c. in mice (3 mice) at day 0 and 14.
A.beta.1-6 peptide was coupled to Q.beta. VLP protein as described
above. Each mice (C57BL/6) was immunized with 10 .mu.g of vaccine
diluted in PBS to 200 .mu.l. Mice were retroorbitally bled on day
21, and the titer of the antibodies specific for the A.beta.1-6
peptide were measured in an ELISA against A.beta.1-6. The
A.beta.1-6 peptide was coupled to bovine RNAse A using the chemical
cross-linker sulfo-SPDP. ELISA plates were coated with coupled
RNAse preparations at a concentration of 10 .mu.g/ml. The plates
were blocked and then incubated with serially diluted mouse sera.
Bound antibodies were detected with enzymatically labeled
anti-mouse IgG antibodies. As a control, preimmune sera of the same
mice were also tested. The results are shown in FIG. 2.
[0352] FIG. 2 shows an ELISA analysis of the IgG
antibodies-specific for A.beta.1-6 peptide in sera of mice
immunized against the A.beta.1-6 peptide coupled to Q.beta. VLP.
The results are shown for the three mice immunized (A1-A3), the
pre-immune serum is indicated as "pre" in the figure; the result
for one pre-immune serum is shown. Comparison of the pre-immune
sera with the sera of mice immunized, with "Qb-Ab-1-6" shows that a
strong specific antibody response against peptide A.beta.1-6 could
be obtained in the absence of adjuvant.
C. ELISA Against A.beta.1-40 Peptide
[0353] Human A.beta. 1-40 or A.beta. 1-42 peptide stock was made in
DMSO and diluted in coating buffer before use. ELISA plates were
coated with 0.1 .mu.g/well A.beta. 1-40 peptide. The plates were
blocked and then incubated with serially diluted mouse serum
obtained above. Bound antibodies were detected with enzymatically
labeled anti-mouse IgG antibody. As a control, sera obtained before
vaccination were also included. The serum dilution showing a mean
three standard deviations above baseline was calculated and defined
as "ELISA titer". No specific antibodies were detected in preimmune
sera. The titer obtained for the three mice was of 1:100000,
showing a strong specific immune response against A.beta. 1-40.
Thus, immunization with A.beta.1-6 coupled to Q.beta. VLP elicits
strong antibody titers cross-reactive with A.beta.1-40.
[0354] FIG. 3 shows the result of the ELISA. The ELISA signal as
the optical density at 405 nm, obtained for the sera of three mice
(A1-A3) immunized with A.beta.1-6 peptide coupled to Q.beta. VLP as
described above, is plotted for each of the dilutions, indicated on
the x-axis. The result for the three mice bled at day 21 is shown.
Also included is a pre-immune serum. The titer of the antibodies in
the sera was determined as described above, and was of 1:100000 for
all three mice.
Example 14
Immunization of Human APP Transgenic Mice
[0355] 8 months old female APP23 mice which carry a human APP
transgene (Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. USA 94:
13287-13292 (1997)) are used for vaccination. The mice are injected
subcutaneously with 25 .mu.g vaccine diluted in sterile PBS and 14
days later boosted with the same amount of vaccine. Mice are bled
from the tail vein before the start of immunization and 7 days
after the booster injection. The sera are analyzed for the presence
of antibodies specific to a A.beta.1-6, to A.beta.1-40 and
A.beta.1-42 by ELISA as described in Example 13.
Example 15
Coupling of Murine A.beta.1-6 to Q.beta. VLP, Injection of the
Vaccine in Mice, and Analysis of the Immune Response
[0356] Murine A.beta.1-6 peptide (sequence: NH2-DAEFGHGGC-CONH2)
(SEQ ID NO: 78) is chemically synthesized, and used for coupling to
Q.beta. VLP as described in Example 13. The vaccine is injected in
C57BL/6 mice, and the titer of the elicited antibodies against
murine A.beta.1-6, murine A.beta.1-40 and murine A.beta.1-42
determined. The immunization and the ELISA determination are
performed as described in Example 13.
Example 16
Binding of Sera Elicited Against A.beta.1-6 to Human APP Transgenic
Mice Plaques and AD Plaques
Immuohistochemistry in Brain Slices
[0357] Consecutive paraffin brain sections of a 18 months, old
heterozygous APP23 mouse and entorhinal cortex sections from an AD
patient Braak Stage III (Institute of Pathology, University Basel)
were used for staining. Antigenicity was enhanced by treating human
brain sections with concentrated formic acid for five minutes and
mouse brain sections by microwave heating at 90.degree. C. for 3
minutes. Mice sera elicited against human A.beta.1-6 (obtained as
described in Example 13) were diluted 1:1000 in PBS with 3% goat
serum and incubated over night. Following rinsing, sections were
incubated for 1 hour with biotinylated anti mouse secondary
antibody diluted 1:200 in PBS. After rinsing, sections were further
processed with the avidin-biotin-peroxidase technique (ABC-Elite
Kit PK6100; Vector Laboratories). Finally, sections were reacted
with Diaminobenzidine (DAB) metal enhanced substrate (Boehringer,
Code 1718096), counterstaind with Hemalum, dehydrated, cleared in
Xylene and coversliped.
[0358] The result of the histologic stains are shown in FIGS. 4 A
and B. Sections were stained with the sera of the three mice
immunized against human A.beta.1-6 coupled to Q.beta. VLP. Each
serum stained positively the amyloid plaques from transgenic mice
and AD. Results for one of the three sera are shown. Sera elicited
against human A.beta.1-6 clearly stain amyloid plaques of the
transgenic human APP23 mouse, as well as amyloid plaques from AD
patients. Pre-immune sera were negative. Extracellular amyloid
plaques and isolated blood vessels are stained by the
antibodies.
Example 17
Specificity of Sera Elicited Against Human A.beta.1-6, Assessed by
Histology of Mice Plaques
Immunohistochemistry in Brain Slices
[0359] Consecutive paraffin brain sections of a 3 months and an 18
months old heterozygous APP23 mouse overexpressing human APP were
stained as described in Example 16 with a representative mouse
serum elicited against human A.beta.1-6 as described in Example 13,
or with a rabbit polyclonal antibody specific for the last 20 amino
acids of murine or human APP and which therefore does not
recognizes A.beta.. The sections incubated with the rabbit
polyclonal antibody were treated as described in Example 16, except
for the use of a biotinylated anti rabbit secondary antibody
(BA1000, Vector Laboratories).
[0360] The result of the histologic stains are shown on FIGS. 5 A,
B, C, D and E. A.beta.1-6, marked on the bottom left of the
sections indicate that sera elicited against A.beta.1-6 have been
used for the staining, while "Pab" indicates that the sections have
been stained with the polyclonal antibody specific for the last 20
amino acids of murine or human APP, corresponding to positions
676-695 in APP695.
[0361] Comparison of the staining of sections from 81 months old
mice (FIGS. 5 A and C) shows that the sera elicited against
A.beta.1-6 do not cross-react with APP expressed in the brain,
which is however stained by the control polyclonal antibody. FIG. 5
B shows a brain section from a 3 months old mouse, a timepoint
where amyloid deposits are not yet visible, stained with the
polyclonal antibody specific for APP. FIGS. 5 D and 5E show a
magnification of the CA1 pyramidal layer of the hippocampus from
FIG. 5A, and FIG. 5B, respectively.
Example 18
A. Coupling of A.beta.1-6 Peptide to fr Capsid Protein
[0362] A solution of 120 .mu.M fr capsid protein in 20 mM Hepes,
150 mM NaCl pH 7:2 is reacted for 30 minutes with a 10 fold molar
excess of SMPH (Pierce), diluted from a stock solution in DMSO, at
25.degree. C. on a rocking shaker. The reaction solution is
subsequently dialyzed twice for 2 hours against 1 L of 20 mM Hepes,
150 mM NaCl, pH 7.2 at 4.degree. C. The dialyzed fr reaction
mixture is then reacted with a a five-fold molar excess of
A.beta.1-6 peptide (sequence: NH2-DAEFRHGGC-CONH2) (SEQ ID NO: 77)
for 2 hours at 16.degree. C. on a rocking shaker. Coupling products
are analysed by SDS-PAGE.
B. Coupling of A.beta.1-6 Peptide to HBcAg-Lys-2cys-Mut
[0363] A solution of 1 ml of 120 uM HBcAg-Lys-2-cys-Mut in 20 mM
Hepes, 150 mM NaCl pH 7.2 is reacted for 30 minutes with a 10 fold
molar excess of SMPH (Pierce), diluted from a stock solution in
DMSO, at 25.degree. C. on a rocking shaker. The reaction solution
is subsequently dialyzed twice for 2 hours against 1 L of 20 mM
Hepes, 1.50 mM NaCl, pH 7.2 at 4.degree. C. The dialyzed
HBcAg-Lys-2cys-Mut reaction mixture is then reacted with a
five-fold molar excess of A.beta.1-6 peptide (sequence:
NH2-DAEFRRGGC-CONH2) (SEQ ID NO: 77) for 2 hours at 16.degree. C.
on a rocking shaker. Coupling products are analysed by
SDS-PAGE.
C. Coupling of A.beta.1-6 Peptide to Pili
[0364] A solution of 125 .mu.M Type-1 pili of E. coli in 20 mM
Hepes, pH 7.4, is reacted for 60 minutes with a 50-fold molar
excess of cross-linker SMPH (Pierce), diluted from a stock solution
in DMSO, at RT on a rocking shaker. The reaction mixture is
desalted on a PD-10 column (Amersham-Pharmacia Biotech). The
protein-containing fractions eluting from the column are pooled,
and the desalted derivatized pili protein is reacted with a
five-fold molar excess of A.beta.1-6 peptide (sequence:
NH2-DAEFRHGGC-CONH2) (SEQ ID NO: 77) for 2 hours at 16.degree. C.
on a rocking shaker. Coupling products are analysed by
SDS-PAGE.
D. Immunization of mice with A.beta.1-6 Peptide Coupled to
fr-Capsid Protein, HBcAg-Lys-2cys-Mut or pili
[0365] A.beta.1-6 peptide coupled to fr-capsid protein,
MBc-Ag-Lys-2cys-Mut or pili as described above is injected s.c. in
mice (3 mice) at day 0 and 14. Each mice (C57BL/6) is immunized
with 10 .mu.g of vaccine diluted in PBS to 200 .mu.l. Mice are
retroorbitally bled on day 21, and the titer of the antibodies
specific for the A.beta.1-6 peptide or A.beta.1-40 or A.beta.1-42
are measured by ELISA as described in Example 13.
Example 19
Immunisation of Rhesus Monkeys with Q.beta.hA.beta.1-6
[0366] In order to test induction of antibodies against human
A.beta. using a human A.beta.1-6 peptide based vaccine in the case
where A.beta.1-6 is a self antigen, rhesus monkeys were immunized
with Q.beta.hA.beta.1-6, as the A.beta. sequence is identical
between humans and Rhesus monkeys. Q.beta.hA.beta.1-6 vaccine was
made as described in Example 13. Four Rhesus monkeys, between 10
and 15 years of age, were immunized at day 0 with 50 .mu.g of
vaccine, and boosted twice at day 28 and 56 with 25 .mu.g of
vaccine. The monkeys were immunized subcutaneously in the back. The
animals were bled at day 0 (prebleed), 42 and 70. 4 ml of blood
were collected from the V. cephalica antebrachii. The titer of
antibodies specific for A.beta.1-40 were measured by ELISA
essentially as described in Example 13, using a secondary antibody
specific for Monkey IgG.
[0367] As humans and rhesus monkeys share the same A.beta.
sequence, the generation of high titer antibodies in rhesus monkeys
specific for A.beta.1-40 shows that immunization with hA.beta.1-6
coupled to Q.beta. breaks tolerance against the self-antigen
A.beta.. Furthermore, antibodies recognizing full length A.beta.
are generated with the coupled A.beta.1-6 fragment in primates.
[0368] The results of the ELISA are shown in FIG. 6. Plotted in the
diagram are the titers of A.beta.1-40 specific antibodies measured
in the sera of the 4 monkeys (1-4) immunized with
Q.beta.hA.beta.1-6 and the average of the titers of the 4 monkeys.
The titers are represented as OD50 titers. OD50 is the dilution of
the antibodies at which the signal reaches half of its maximal
value. The maximal value (OD max) was obtained from a reference
serum originating from a monkey immunised with Q.beta.hA.beta.1-27
and recognizing A.beta.1-40 as well, and measured on the same ELISA
plate.
[0369] Two monkeys (described above) were bled at day 97, 110, 117,
124, 138, 143, 152, 159, 166, and received a third boost with 25
.mu.g of vaccine at day 110. Sera were pooled (99 ml) and used for
affinity purification of A.beta.1-6-specific antibodies. These
antibodies were used for immunohistochemical staining at a
concentration of 1.5 .mu.g/ml and a biotinylated secondary
anti-monkey antibody was used for detection. Paraffine brain
sections of 18 months old heterozygous APP23 mouse and an AD
patient--Braak Stage III--were used for staining. Plaque-specific
staining was observed both in APP23 mouse brain sections and in the
AD patient brain sections (FIG. 7).
[0370] The result of the histological analysis is shown in FIGS. 7
A and B. Depicted in FIG. 7 A is the staining of human APP
transgenic mouse plaques (APP23 strain) with the above described
affinity purified antiserum specific for A.beta.1-6. FIG. 7 B shows
the staining of human AD plaques with the same purified antiserum.
The purified antiserum was used at a concentration of 1.5 .mu.g/ml
in both cases. Typical plaques are indicated by an arrow on both
figures.
Example 20
Coupling of Marine A.beta.1-6 to AP205 VLP, Immunisation of Mice
and Analysis of Immune Response
A. Coupling of Murine A.beta.1-6 Peptide to AP205 VLP
[0371] The peptide murine A.beta.1-6 (mA.beta.1-6, sequence:
NH2-DAEFGHGGC-CONH2 (SEQ ID NO: 78) was chemically synthesized; the
initial NH2 group indicates that the peptide has a tree N-terminus,
and the terminal NH2 group indicates that the peptide has an
amidated carboxy-terminus). AP205 VLP (expressed and purified as
described in Example 1), in 20 mM Hepes, 150 mM NaCl, pH 8.0 (HBS,
pH 8.0) was reacted at a concentration of 2 mg/ml (determined in a
Bradford assay), with 2.86 mM SMPH (Pierce, Rockford Ill.), diluted
from a 100 mM stock in DMSO, for 30 minutes at room temperature
(RT). The reaction mixture was then dialyzed twice against a
1000-fold volume of HBS, pH 7.4, at 4.degree. C. for two hours; the
resulting dialyzed and derivatized AP205 VLP was flash frozen in
liquid nitrogen and stored at -20.degree. C. overnight. Derivatized
AP205 VLP was diluted with one volume of 20 mM HBS, pH 7.4, and
reacted 2 hours at 15.degree. C. under shaking with 719 .mu.M
mA.beta.1-6 peptide diluted in the reaction mixture from a 50 mM
stock in DMSO. The coupling reaction was dialyzed twice against a
1000-fold volume HBS, pH 7.4, for 2 hours and overnight. The
dialyzed reaction mixture was flash frozen in liquid nitrogen in
aliquots for storage at -80.degree. C. until further use.
[0372] An aliquot was thawed, and coupling of the mA.beta.1-6
peptide to the AP205 VLP subunits assessed by SDS-PAGE and the
protein concentration measured in a Bradford assay. The result of
the coupling reaction is shown in FIG. 8.
[0373] FIG. 8 shows the SDS-PAGE analysis of the coupling reaction
of mA.beta.1-6 peptide to AP205 VLP. The samples were run under
reducing conditions on a 16% Tris-glycine gel and stained with
coomassie brilliant blue. Lane 1 is the protein marker, with
corresponding molecular weights indicated on the left border of the
gel; lane 2, AP205 VLP protein; lane 3, derivatized AP205 VLP; lane
4, the supernatant of the coupling reaction of AP205 VLP to
mA.beta.1-6 peptide; lane 5, the pellet of the coupling reaction of
AP205 VLP to mA.beta.1-6 peptide. No AP205 VLP subunits left
uncoupled could be detected on the gel while bands corresponding to
several peptides per subunits were visible, demonstrating a very
high coupling efficiency. In particular, there is much more than
one A.beta.1-6 peptide per AP205 VLP subunit.
B. Immunisation of Mice with mA.beta.1-6 Peptide Coupled to AP205
VLP and Analysis of Immune Response
[0374] AP205 VLP coupled to mA.beta.1-6 peptide was injected s.c.
in mice (3 msec) at day 0 and 14, mA.beta.1-6 peptide was coupled
to AP205 VLP as described above. Each mice (C57BL/6) was immunized
with 25 .mu.g of vaccine diluted in PBS to 200 .mu.l. Mice were
retroorbitally bled on day 21, and the titer of the antibodies
specific for the mA.beta.1-6 peptide were measured in an ELISA
against mA.beta.1-6. The mA.beta.1-6 peptide was coupled to bovine
RNAse A using the chemical cross-linker sulfo-SPDP. ELISA plates
were coated with preparations of RNAse-mA.beta.1-6 at a
concentration of 10 .mu.g/ml. The plates were blocked and then
incubated with serially diluted mouse sera. Bound antibodies were
detected with enzymatically labeled anti-mouse IgG antibodies. As a
control, preimmune sera of the same mice were also tested. The
results are shown in FIG. 9.
[0375] FIG. 9 shows an ELISA analysis of the IgG antibodies
specific for mA.beta.1-6 peptide in sera of mice immunized with the
mA.beta.1-6 peptide coupled to AP205 VLP. The results are shown for
the sera of the three immunized mice collected at day 21 (A1 d21-A3
d21), the pre-immune serum is indicated as "pre imm" in the figure;
the result for one pre-immune serum is shown. Comparison of the
pre-immune serum with the sera of the mice immunized with
mA.beta.1-6 coupled to AP205 VLP shows that a strong specific
antibody response against peptide mA.beta.1-6, which is a
self-antigen, could be obtained in the absence of adjuvant.
Furthermore, coupling of a self-peptide to AP205 VLP leads to break
of tolerance against this peptide, and to a very high specific
immune response. Thus, AP205 VLP is suitable for generating high
antibody titers against A.beta. peptides in the absence of
adjuvant.
Example 21
Immunisation with Q.beta.hA.beta.1-6 Reduces Amyloid Plaques in
Transgenic Mice Over-Expressing the "Swedish/London" Mutant Amyloid
Precursor Protein
[0376] This example demonstrates that immunization with
Q.beta.hA.beta.1-6 in a mouse model developing Alzheimer's
disease-like diffuse (Congo-Red negative) amyloid plaques, resulted
in a massive reduction of plaque density in neocortical and
subcortical brain areas. Histological occurrence of diffuse amyloid
plaques is a prominent feature of AD brain pathology (Selkoe, 1994,
Annu. Rev. Neurosci. 17: 489-517) and, therefore, the example
demonstrates that immunization with Q.beta.hA.beta.1-6 provides an
effective approach for the treatment of Alzheimer's disease.
[0377] To evaluate the therapeutic efficacy of immunization with
Q.beta.-A.beta.1-6 transgenic mice over-expressing the
"Swedish/London" mutant amyloid precursor protein under the control
of the mouse Thy-1 promoter (APP24; K670N/M671L; V717I, patent No.
WO980-36-4423) were used. This mouse strain is characterized by a
large number amyloid plaques in the neocortex, hippocampus, caudate
putamen, and thalamus at an age of 18 months. Plaques can be first
observed at an age of 9 months. Histologically, the amyloid plaques
in APP24 mice are predominantly of a diffuse type, i.e. they are
negative in Congo-Red staining. To a lesser degree, also compact
amyloid plaques (Congo-Red positive) can be found.
[0378] Human A.beta.1-6 peptide coupled to Q.beta. VLP
(Q.beta.hA.beta.1-6) was made as described in Example 13. In terms
of the experimental procedure followed, which is not necessary for
describing or enabling the invention, APP24 transgenic mice 9.5
months of age were injected subcutaneously at day 0 with 25 .mu.g
of Q.beta.hA.beta.1-6 in phosphate-buffered saline (PBS)
(administered as 2.times.100 .mu.l per mouse) (n=16) or as negative
controls with PBS (administered as 2.times.100 .mu.l per mouse)
(n=9) or with Q.beta. virus-like particle devoid of coupled antigen
(n=11). Mice were subsequently injected 25 .mu.g of
Q.beta.hA.beta.1-6-vaccine, Q.beta., or PBS on day 15, 49, 76, 106,
140, 169, 200, 230, 259, and 291. Animals were bled 1-2 days before
the first immunization (day 0) and on day 56, 90, 118, 188, 214,
246, and 272 via the tail vein. Blood serum was also collected on
day 305, at which time also brains were collected for
histopathology (age of the mice at this time point: 19.5
months).
[0379] The titer of antibodies specific for A.beta.1-40 were
measured by ELISA essentially as described in Example 13. The
results of the ELISA are shown in FIG. 10. Plotted in the diagram
are the titers of A.beta.1-40 or A.beta.1-42 specific antibodies
measured in the sera of mice immunized with Q.beta.hA.beta.1-6. The
titers are represented as OD50% titers. OD50% is the dilution of
the antibodies at which the signal reaches half of its maximal
value. The maximal value (OD max) was obtained from a reference
antibody recognizing A.beta.1-40 and Ab42, and measured on the same
ELISA plate. All Q.beta.hA.beta.1-6 immunized mice developed OD50%
titers above 1:8000 (pre-immune serum titers were below 1:100)
demonstrating a consistent antibody response to Q.beta.hA.beta.1-6
even in old APP24 mice (FIG. 10). Median OD50% titers in the
immunized group were in the range of 1:20,000 to 1:50,000
throughout the immunization period.
[0380] For quantification of amyloid plaques, brains. Were fixed by
immersion in 4% formaldehyde in 0.1 M PBS at 4.degree. C. After
dehydration with ethanol, brains were embedded in paraffin and cut
sagitally with a microtome at 4 .mu.m thickness. Sections were
mounted onto super frost slides and dried at 37.degree. C. Sections
were washed on PBS and antigenicity enhanced by microwave heating
at 90.degree. C. for 3 minutes in 0.1 M citric acid buffer. NT11
antisera (anti A.beta.1-40. Sturchler-Pierrat et al., 1997, Proc.
Natl. Acad. Sci. 94: 13287-13292) were diluted 1:1000 in PBS with
3% goat serum and incubated over night at 4.degree. C. Following
rinsing, sections were incubated for 1 hour with biotinylated anti
rabbit IgG secondary antibody (BA1000, Vector Laboratories) diluted
1:200 in PBS. After rinsing, sections were further processed with
the avidin-biotin-peroxidase technique (ABC-Elite Kit PK6100;
Vector Laboratories). Finally, sections were reacted with
Diaminobenzidine (DAB) metal enhanced substrate (Boehringer, Code
1718096), counterstained with Hemalum, dehydrated, cleared, in
Xylene and cover slipped. Systematic-random series of brain
sections at three different anatomical planes per animal were used
for the analysis. Amyloid plaques were quantified using an MCID
image analyzer (Imaging Research, Brock University, Ontario-Canada,
Program Version M5 elite). The microscopic image was digitized by
use of a Xillix black and white CCD TV camera and stored with
640.times.480 pixel resolution at 256 gray levels. The pixel size
was calibrated using an object micrometer at 5.times.magnification
(Leica Neoplan Objective). Using a motor driven microscope stage
for exact positioning of adjacent object fields the entire
neocortex and olfactory nucleus of each section was analysed. For
each object field the anatomical area was defined by manual
outline. For each individual section the sample area was defined by
manual threshold setting (grey level) between immunopositive
amyloid plaques and tissue background. Isolated tissue artifacts
were excluded by manual outline. Raw data are measured as
individual counts (amyloid deposits) and proportional area values
(immunopositive amyloid/cortex or olfactory nucleus).
[0381] Data of each mouse were normalized to number of deposits
(plaques) per mm.sup.2 and total plaque area in % of the entire
neocortex. Q.beta.hA.beta.1-6 immunized mice revealed a dramatic
reduction of amyloid deposits in the cortex and subcortical areas
as compared to either PBS or Q.beta. injected control groups (FIG.
11). Both the median number of deposits and the total plaque area
were highly significantly reduced between 80-98 % compared to the
PBS group in the cortex, caudate putamen, hippocampus, and thalamus
(p<0.001 vs. PBS-group, Mann-Whitney test; FIG. 12 ).
[0382] In a second study, APP24 transgenic mice 13.5 months of age
were injected subcutaneously at day 0 with 25 .mu.g of
Q.beta.hA.beta.1-6 in phosphate-buffered saline (PBS) (administered
as 2.times.100 .mu.l per mouse) (n=15) or as negative controls with
PBS (administered as 2.times.100 .mu.l per mouse) (n=15). Mice were
subsequently injected 25 .mu.g of Q.beta.hA.beta.1-6-vaccine, or
PBS on day 16, 46, 76, 109, 140, and 170. Animals were bled 1-2
days before the first immunization (day 0) and on day 31, 59, 128,
and 154 via the tail vein. Blood serum was also collected on day
184, at which time also brains were collected for histopathology
(age of the mice at this time point: 19.5 months). The titer of
antibodies specific for A.beta.1-40 were determined and expressed
as described above and again all immunized mice were found to
respond to Q.beta.hA.beta.1-6 immunization with serum OD50% titers
at least above 1:2000 (not shown). Median OD50% titers were in the
range of 1:10,000 to 1:50,000 throughout the immunization period.
Quantification of amyloid deposits was done as described above.
Compared to the experiment where immunization was initiated earlier
(i.e. at an age of 9.5 months) the reduction of plaque deposit
number (-55%) and area (-32 %) was less dramatic in the neocortex,
but still very pronounced (FIG. 13) and highly significant
(p>0.001 vs. PBS, Mann-Whitney test). In subcortical areas
plaque deposit number and area were reduced by 60-90% in the to
Q.beta.hA.beta.1-6 immunized group. The more pronounced effect in
these areas as compared to the cortex is probably related to the
more protracted time course of plaque formation in these areas.
[0383] Taken together, both experiments demonstrate that
Q.beta.hA.beta.1-6 immunization in transgenic mice over-expressing
the "Swedish/London" mutant amyloid precursor protein dramatically
reduces the occurrence of amyloid deposits in these mice.
[0384] FIG. 10: Serum anti A.beta.40/42 antibody titers (OD50%) in
transgenic mice over-expressing the "Swedish/London" mutant amyloid
precursor protein. Mice were immunized with Q.beta.hA.beta.1-6
between 9.5 and 19 months of age. Shown are individual values
(black dots) and box plots, where the ends of the boxes define the
25.sup.th and 75.sup.th percentiles, with a line at the median and
error bars defining the 10.sup.th and 90.sup.th percentiles
(outlyers are shown as dots).
[0385] FIG. 11: Immunhistochemical staining of amyloid plaques in
sagittal brain sections. The sagittal brain section of a transgenic
mouse over-expressing the "Swedish/London" mutant amyloid precursor
protein immunized with Q.beta. (A) or Q.beta.hA.beta.1-6 (B)
vaccine is shown in the Figure.
[0386] FIG. 12: Quantification of plaque deposition in transgenic
mice over-expressing the "Swedish/London" mutant amyloid precursor
protein after immunization between 9.5 and 19 months of age. (A)
Cortical plaque density. (B) Cortical plaque area. (C) Plaque
density in the caudate putamen. (D) Plaque area in the caudate
putamen. (E) Plaque density in the hippocampus. (F) Plaque area in
the hippocampus. (G) Plaque density in the thalamus. (H) Plaque
area in the thalamus. Plaque density is expressed in
plaques/mm.sup.2, plaque area in percent of tissue area covered by
amyloid beta. Data are shown as individual values (black dots) and
box plot. The ends of the boxes define the 25.sup.th and 75.sup.th
percentiles, with a line at the median and error bars defining the
10.sup.th and 90.sup.th percentiles. ** p<0.001 (Mann Whitney
Rank Sum Test), PBS, n=9, Q.beta., n=11, Q.beta.hA.beta.1-6,
n=16.
Example 22
Immunisation with Q.beta.hAB.beta.1-6 B Reduces Amyloid Plaques in
Transgenic Mice Over-Expressing the "Swedish" Mutant Amyloid
Precursor Protein
[0387] This example demonstrates that immunization with
Q.beta.hA.beta.1-6 provides an effective approach for the treatment
of Alzheimer's disease even when the immunization is initiated in a
very advanced stage of amyloid plaque pathology. The amyloid plaque
deposition process in the AD mouse model used in this example
starts already at an age about 6 months (Sturchler-Pierrat et al.,
1997, Proc. Natl. Acad. Sci. 94: 13287-13292). In the study
described herein, immunization with Q.beta.hA.beta.1-6 was
initiated at an age of 18 months, where already a high number of
compact plaques had been formed in the cortex. The example also
demonstrates the ability of Q.beta.hA.beta.1-6 to induce
A.beta.40/42 antibodies in very aged animals (no non-responders in
19 immunized mice).
[0388] To evaluate the therapeutic effects of immunization with
Q.beta.hA.beta.1-6 transgenic mice over-expressing the "Swedish"
mutant amyloid precursor protein (APP23; K670N/M671L,
Sturchler-Pierrat et al., 1997, Proc. Natl. Acad. Sci. 94:
13287-13292) were used. The Alzheimer's-like pathology in these
mice has been extensively characterized (Calhoun et al., 1998,
Nature 395: 755-756; Phinney et al., 1999, J. Neurosci 19:
8552-8559; Bondolfi et al., 2002, J. Neurosci. 22: 515-522).
[0389] Human A.beta.1-6 peptide coupled to Q.beta. VLP
(Q.beta.hA.beta.1-6) was made as described in Example 13. In terms
of the experimental procedure followed, which is not necessary for
describing or enabling the invention, APP23 transgenic mice 18
months of age were injected subcutaneously at day 0 with 25 .mu.g
of Q.beta.hA.beta.1-6 dissolved in phosphate-buffered saline
(administered as 2.times.100 .mu.l per mouse) (n=19) or
phosphate-buffered saline as a negative control (n=17) and boosted
on day 13, 27-34, 61-63, 90-96, and 123-130 with 25 .mu.g of
vaccine. Animals were bled 1-2 days before the first immunization
(day 0) and on day 41-45, and day 68 via the tail vein. Blood serum
was also collected on day 152-154, at which time also brains were
collected for histopathology (age of the mice at this time point:
23 months).
[0390] The titer of antibodies specific for A.beta.1-40 were
measured by ELISA essentially as described in Example 13 and the
results expressed as described in Example 21. The results of the
ELISA are shown in FIG. 14. All Q.beta.hA.beta.1-6 immunized mice
developed OD50% titers above 1:2000 (pre-immune serum titers were
below 1:100) demonstrating a consistent antibody response to
Q.beta.-A.beta.1-6 even in very old mice (FIG. 14). Median OD50%
titers were in the range of 1:9,000 to 1:20,000 throughout the
immunization period.
[0391] Quantification of amyloid plaques was done as described in
Example 21. Data of each mouse were normalized to number of
deposits (plaques) per mm.sup.2 and total plaque area in % of the
entire neocortex. Q.beta.hA.beta.1-6 immunized mice revealed a
smaller number of deposits in the cortex (FIG. 15, FIG. 16), mostly
due to a reduction of small sized plaques. Compared to the
non-immunized group the median plaque number was reduced by 33% in
the Q.beta.hA.beta.1-6 immunized group (p<0.001 vs PBS-group,
Mann-Whitney test). Since mostly small-sized plaques were affected
the reduction of the total plaque area was moderate and amounted to
10% (p<0.01 vs. PBS group, Mann-Whitney test).
[0392] FIG. 14: Serum anti A.beta.40/42 antibody titers (OD50%) in
transgenic mice over-expressing the "Swedish" mutant amyloid
precursor protein. Mice were immunized with Q.beta.hA.beta.1-6
between 18 and 23 months of age. Shown are individual values (black
dots) and box plots, where the ends of the boxes define the
25.sup.th and 75.sup.th percentiles, with a line at the median and
error bars defining the 10.sup.th and 90.sup.th percentiles
(outlyers are shown as dots).
[0393] FIG. 15: Immunhistochemical staining of amyloid plaques. In
sagittal brain sections. Arrows point to small sized deposits.
Shown in the Figure is a sagittal brain section from a transgenic
mouse over-expressing the "Swedish" mutant amyloid precursor
protein immunized with PBS (A) or Q.beta.-A.beta.1-6 (B).
[0394] FIG. 16: Quantification of plaque deposition in transgenic
mice over-expressing the "Swedish" mutant amyloid precursor protein
after immunization between 18 and 23 months of age. (A) Cortical
plaque density. (B) Cortical plaque area. Plaque density is
expressed in plaques/mm.sup.2, plaque area in percent of tissue
area covered by amyloid beta. Data are shown as individual values
(black dots) and box plot. The ends of the boxes define the
25.sup.th and 75.sup.th percentiles, with a line at the median and
error bars defining the 10.sup.th and 90.sup.th percentiles. **
p<0.001 (Mann Whitney Rank Sum Test). PBS, n=17,
Q.beta.hA.beta.1-6, n=19.
[0395] Having now fully described the present invention in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious to one of ordinary skill in
the art that the same can be performed by modifying or changing the
invention within a wide and equivalent range of conditions,
formulations and other parameters without affecting the scope of
the invention or any specific embodiment thereof, and that such
modifications or changes are intended to be encompassed within the
scope of the appended claims.
[0396] All publications, patents and patent applications mentioned
in this specification are indicative of the level of skill of those
skilled in the art to which this invention pertains, and are herein
incorporated by reference to the same extent as if each individual
publication, patent or patent application was specifically and
individually indicated to be incorporated by reference.
Sequence CWU 1
1
931172PRTEscherichia coli 1Met Ala Val Val Ser Phe Gly Val Asn Ala
Ala Pro Thr Thr Pro Gln1 5 10 15Gly Gln Gly Arg Val Thr Phe Asn Gly
Thr Val Val Asp Ala Pro Cys 20 25 30Ser Ile Ser Gln Lys Ser Ala Asp
Gln Ser Ile Asp Phe Gly Gln Leu 35 40 45Ser Lys Ser Phe Leu Ala Asn
Asp Gly Gln Ser Lys Pro Met Asn Leu 50 55 60Asp Ile Glu Leu Val Asn
Cys Asp Ile Thr Ala Phe Lys Asn Gly Asn65 70 75 80Ala Lys Thr Gly
Ser Val Lys Leu Ala Phe Thr Gly Pro Thr Val Ser 85 90 95Gly His Pro
Ser Glu Leu Ala Thr Asn Gly Gly Pro Gly Thr Ala Ile 100 105 110Met
Ile Gln Ala Ala Gly Lys Asn Val Pro Phe Asp Gly Thr Glu Gly 115 120
125Asp Pro Asn Leu Leu Lys Asp Gly Asp Asn Val Leu His Tyr Thr Thr
130 135 140Val Gly Lys Lys Ser Ser Asp Gly Asn Ala Gln Ile Thr Glu
Gly Ala145 150 155 160Phe Ser Gly Val Ala Thr Phe Asn Leu Ser Tyr
Gln 165 1702182PRTEscherichia coli 2Met Lys Ile Lys Thr Leu Ala Ile
Val Val Leu Ser Ala Leu Ser Leu1 5 10 15Ser Ser Thr Ala Ala Leu Ala
Ala Ala Thr Thr Val Asn Gly Gly Thr 20 25 30Val His Phe Lys Gly Glu
Val Val Asn Ala Ala Cys Ala Val Asp Ala 35 40 45Gly Ser Val Asp Gln
Thr Val Gln Leu Gly Gln Val Arg Thr Ala Ser 50 55 60Leu Ala Gln Glu
Gly Ala Thr Ser Ser Ala Val Gly Phe Asn Ile Gln65 70 75 80Leu Asn
Asp Cys Asp Thr Asn Val Ala Ser Lys Ala Ala Val Ala Phe 85 90 95Leu
Gly Thr Ala Ile Asp Ala Gly His Thr Asn Val Leu Ala Leu Gln 100 105
110Ser Ser Ala Ala Gly Ser Ala Thr Asn Val Gly Val Gln Ile Leu Asp
115 120 125Arg Thr Gly Ala Ala Leu Thr Leu Asp Gly Ala Thr Phe Ser
Ser Glu 130 135 140Thr Thr Leu Asn Asn Gly Thr Asn Thr Ile Pro Phe
Gln Ala Arg Tyr145 150 155 160Phe Ala Thr Gly Ala Ala Thr Pro Gly
Ala Ala Asn Ala Asp Ala Thr 165 170 175Phe Lys Val Gln Tyr Gln
1803853DNAEscherichia coli 3acgtttctgt ggctcgacgc atcttcctca
ttcttctctc caaaaaccac ctcatgcaat 60ataaacatct ataaataaag ataacaaata
gaatattaag ccaacaaata aactgaaaaa 120gtttgtccgc gatgctttac
ctctatgagt caaaatggcc ccaatgtttc atcttttggg 180ggaaactgtg
cagtgttggc agtcaaactc gttgacaaac aaagtgtaca gaacgactgc
240ccatgtcgat ttagaaatag ttttttgaaa ggaaagcagc atgaaaatta
aaactctggc 300aatcgttgtt ctgtcggctc tgtccctcag ttctacgacg
gctctggccg ctgccacgac 360ggttaatggt gggaccgttc actttaaagg
ggaagttgtt aacgccgctt gcgcagttga 420tgcaggctct gttgatcaaa
ccgttcagtt aggacaggtt cgtaccgcat cgctggcaca 480ggaaggagca
accagttctg ctgtcggttt taacattcag ctgaatgatt gcgataccaa
540tgttgcatct aaagccgctg ttgccttttt aggtacggcg attgatgcgg
gtcataccaa 600cgttctggct ctgcagagtt cagctgcggg tagcgcaaca
aacgttggtg tgcagatcct 660ggacagaacg ggtgctgcgc tgacgctgga
tggtgcgaca tttagttcag aaacaaccct 720gaataacgga accaatacca
ttccgttcca ggcgcgttat tttgcaaccg gggccgcaac 780cccgggtgct
gctaatgcgg atgcgacctt caaggttcag tatcaataac ctacctaggt
840tcagggacgt tca 8534132PRTBacteriophage Q-beta 4Ala Lys Leu Glu
Thr Val Thr Leu Gly Asn Ile Gly Lys Asp Gly Lys1 5 10 15Gln Thr Leu
Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val 20 25 30Ala Ser
Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg Val 35 40 45Thr
Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys Val 50 55
60Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser Cys65
70 75 80Asp Pro Ser Val Thr Arg Gln Ala Tyr Ala Asp Val Thr Phe Ser
Phe 85 90 95Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg Thr
Glu Leu 100 105 110Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp Ala
Ile Asp Gln Leu 115 120 125Asn Pro Ala Tyr 1305329PRTBacteriophage
Q-beta 5Met Ala Lys Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Lys Asp
Gly1 5 10 15Lys Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr
Asn Gly 20 25 30Val Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu
Glu Lys Arg 35 40 45Val Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg
Lys Asn Tyr Lys 50 55 60Val Gln Val Lys Ile Gln Asn Pro Thr Ala Cys
Thr Ala Asn Gly Ser65 70 75 80Cys Asp Pro Ser Val Thr Arg Gln Ala
Tyr Ala Asp Val Thr Phe Ser 85 90 95Phe Thr Gln Tyr Ser Thr Asp Glu
Glu Arg Ala Phe Val Arg Thr Glu 100 105 110Leu Ala Ala Leu Leu Ala
Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln 115 120 125Leu Asn Pro Ala
Tyr Trp Thr Leu Leu Ile Ala Gly Gly Gly Ser Gly 130 135 140Ser Lys
Pro Asp Pro Val Ile Pro Asp Pro Pro Ile Asp Pro Pro Pro145 150 155
160Gly Thr Gly Lys Tyr Thr Cys Pro Phe Ala Ile Trp Ser Leu Glu Glu
165 170 175Val Tyr Glu Pro Pro Thr Lys Asn Arg Pro Trp Pro Ile Tyr
Asn Ala 180 185 190Val Glu Leu Gln Pro Arg Glu Phe Asp Val Ala Leu
Lys Asp Leu Leu 195 200 205Gly Asn Thr Lys Trp Arg Asp Trp Asp Ser
Arg Leu Ser Tyr Thr Thr 210 215 220Phe Arg Gly Cys Arg Gly Asn Gly
Tyr Ile Asp Leu Asp Ala Thr Tyr225 230 235 240Leu Ala Thr Asp Gln
Ala Met Arg Asp Gln Lys Tyr Asp Ile Arg Glu 245 250 255Gly Lys Lys
Pro Gly Ala Phe Gly Asn Ile Glu Arg Phe Ile Tyr Leu 260 265 270Lys
Ser Ile Asn Ala Tyr Cys Ser Leu Ser Asp Ile Ala Ala Tyr His 275 280
285Ala Asp Gly Val Ile Val Gly Phe Trp Arg Asp Pro Ser Ser Gly Gly
290 295 300Ala Ile Pro Phe Asp Phe Thr Lys Phe Asp Lys Thr Lys Cys
Pro Ile305 310 315 320Gln Ala Val Ile Val Val Pro Arg Ala
3256129PRTBacteriophage R17 6Ala Ser Asn Phe Thr Gln Phe Val Leu
Val Asn Asp Gly Gly Thr Gly1 5 10 15Asn Val Thr Val Ala Pro Ser Asn
Phe Ala Asn Gly Val Ala Glu Trp 20 25 30Ile Ser Ser Asn Ser Arg Ser
Gln Ala Tyr Lys Val Thr Cys Ser Val 35 40 45Arg Gln Ser Ser Ala Gln
Asn Arg Lys Tyr Thr Ile Lys Val Glu Val 50 55 60Pro Lys Val Ala Thr
Gln Thr Val Gly Gly Val Glu Leu Pro Val Ala65 70 75 80Ala Trp Arg
Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe Ala 85 90 95Thr Asn
Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu Leu 100 105
110Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly Ile
115 120 125Tyr7130PRTBacteriophage fr 7Met Ala Ser Asn Phe Glu Glu
Phe Val Leu Val Asp Asn Gly Gly Thr1 5 10 15Gly Asp Val Lys Val Ala
Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30Trp Ile Ser Ser Asn
Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45Val Arg Gln Ser
Ser Ala Asn Asn Arg Lys Tyr Thr Val Lys Val Glu 50 55 60Val Pro Lys
Val Ala Thr Gln Val Gln Gly Gly Val Glu Leu Pro Val65 70 75 80Ala
Ala Trp Arg Ser Tyr Met Asn Met Glu Leu Thr Ile Pro Val Phe 85 90
95Ala Thr Asn Asp Asp Cys Ala Leu Ile Val Lys Ala Leu Gln Gly Thr
100 105 110Phe Lys Thr Gly Asn Pro Ile Ala Thr Ala Ile Ala Ala Asn
Ser Gly 115 120 125Ile Tyr 1308130PRTBacteriophage GA 8Met Ala Thr
Leu Arg Ser Phe Val Leu Val Asp Asn Gly Gly Thr Gly1 5 10 15Asn Val
Thr Val Val Pro Val Ser Asn Ala Asn Gly Val Ala Glu Trp 20 25 30Leu
Ser Asn Asn Ser Arg Ser Gln Ala Tyr Arg Val Thr Ala Ser Tyr 35 40
45Arg Ala Ser Gly Ala Asp Lys Arg Lys Tyr Ala Ile Lys Leu Glu Val
50 55 60Pro Lys Ile Val Thr Gln Val Val Asn Gly Val Glu Leu Pro Gly
Ser65 70 75 80Ala Trp Lys Ala Tyr Ala Ser Ile Asp Leu Thr Ile Pro
Ile Phe Ala 85 90 95Ala Thr Asp Asp Val Thr Val Ile Ser Lys Ser Leu
Ala Gly Leu Phe 100 105 110Lys Val Gly Asn Pro Ile Ala Glu Ala Ile
Ser Ser Gln Ser Gly Phe 115 120 125Tyr Ala 1309132PRTBacteriophage
SP 9Met Ala Lys Leu Asn Gln Val Thr Leu Ser Lys Ile Gly Lys Asn
Gly1 5 10 15Asp Gln Thr Leu Thr Leu Thr Pro Arg Gly Val Asn Pro Thr
Asn Gly 20 25 30Val Ala Ser Leu Ser Glu Ala Gly Ala Val Pro Ala Leu
Glu Lys Arg 35 40 45Val Thr Val Ser Val Ala Gln Pro Ser Arg Asn Arg
Lys Asn Phe Lys 50 55 60Val Gln Ile Lys Leu Gln Asn Pro Thr Ala Cys
Thr Arg Asp Ala Cys65 70 75 80Asp Pro Ser Val Thr Arg Ser Ala Phe
Ala Asp Val Thr Leu Ser Phe 85 90 95Thr Ser Tyr Ser Thr Asp Glu Glu
Arg Ala Leu Ile Arg Thr Glu Leu 100 105 110Ala Ala Leu Leu Ala Asp
Pro Leu Ile Val Asp Ala Ile Asp Asn Leu 115 120 125Asn Pro Ala Tyr
13010329PRTBacteriophage SP 10Ala Lys Leu Asn Gln Val Thr Leu Ser
Lys Ile Gly Lys Asn Gly Asp1 5 10 15Gln Thr Leu Thr Leu Thr Pro Arg
Gly Val Asn Pro Thr Asn Gly Val 20 25 30Ala Ser Leu Ser Glu Ala Gly
Ala Val Pro Ala Leu Glu Lys Arg Val 35 40 45Thr Val Ser Val Ala Gln
Pro Ser Arg Asn Arg Lys Asn Phe Lys Val 50 55 60Gln Ile Lys Leu Gln
Asn Pro Thr Ala Cys Thr Arg Asp Ala Cys Asp65 70 75 80Pro Ser Val
Thr Arg Ser Ala Phe Ala Asp Val Thr Leu Ser Phe Thr 85 90 95Ser Tyr
Ser Thr Asp Glu Glu Arg Ala Leu Ile Arg Thr Glu Leu Ala 100 105
110Ala Leu Leu Ala Asp Pro Leu Ile Val Asp Ala Ile Asp Asn Leu Asn
115 120 125Pro Ala Tyr Trp Ala Ala Leu Leu Val Ala Ser Ser Gly Gly
Gly Asp 130 135 140Asn Pro Ser Asp Pro Asp Val Pro Val Val Pro Asp
Val Lys Pro Pro145 150 155 160Asp Gly Thr Gly Arg Tyr Lys Cys Pro
Phe Ala Cys Tyr Arg Leu Gly 165 170 175Ser Ile Tyr Glu Val Gly Lys
Glu Gly Ser Pro Asp Ile Tyr Glu Arg 180 185 190Gly Asp Glu Val Ser
Val Thr Phe Asp Tyr Ala Leu Glu Asp Phe Leu 195 200 205Gly Asn Thr
Asn Trp Arg Asn Trp Asp Gln Arg Leu Ser Asp Tyr Asp 210 215 220Ile
Ala Asn Arg Arg Arg Cys Arg Gly Asn Gly Tyr Ile Asp Leu Asp225 230
235 240Ala Thr Ala Met Gln Ser Asp Asp Phe Val Leu Ser Gly Arg Tyr
Gly 245 250 255Val Arg Lys Val Lys Phe Pro Gly Ala Phe Gly Ser Ile
Lys Tyr Leu 260 265 270Leu Asn Ile Gln Gly Asp Ala Trp Leu Asp Leu
Ser Glu Val Thr Ala 275 280 285Tyr Arg Ser Tyr Gly Met Val Ile Gly
Phe Trp Thr Asp Ser Lys Ser 290 295 300Pro Gln Leu Pro Thr Asp Phe
Thr Gln Phe Asn Ser Ala Asn Cys Pro305 310 315 320Val Gln Thr Val
Ile Ile Ile Pro Ser 32511130PRTBacteriophage MS2 11Met Ala Ser Asn
Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr1 5 10 15Gly Asp Val
Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30Trp Ile
Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45Val
Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55
60Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val65
70 75 80Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile
Phe 85 90 95Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln
Gly Leu 100 105 110Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala
Ala Asn Ser Gly 115 120 125Ile Tyr 13012133PRTBacteriophage M11
12Met Ala Lys Leu Gln Ala Ile Thr Leu Ser Gly Ile Gly Lys Lys Gly1
5 10 15Asp Val Thr Leu Asp Leu Asn Pro Arg Gly Val Asn Pro Thr Asn
Gly 20 25 30Val Ala Ala Leu Ser Glu Ala Gly Ala Val Pro Ala Leu Glu
Lys Arg 35 40 45Val Thr Ile Ser Val Ser Gln Pro Ser Arg Asn Arg Lys
Asn Tyr Lys 50 55 60Val Gln Val Lys Ile Gln Asn Pro Thr Ser Cys Thr
Ala Ser Gly Thr65 70 75 80Cys Asp Pro Ser Val Thr Arg Ser Ala Tyr
Ser Asp Val Thr Phe Ser 85 90 95Phe Thr Gln Tyr Ser Thr Val Glu Glu
Arg Ala Leu Val Arg Thr Glu 100 105 110Leu Gln Ala Leu Leu Ala Asp
Pro Met Leu Val Asn Ala Ile Asp Asn 115 120 125Leu Asn Pro Ala Tyr
13013133PRTBacteriophage MX1 13Met Ala Lys Leu Gln Ala Ile Thr Leu
Ser Gly Ile Gly Lys Asn Gly1 5 10 15Asp Val Thr Leu Asn Leu Asn Pro
Arg Gly Val Asn Pro Thr Asn Gly 20 25 30Val Ala Ala Leu Ser Glu Ala
Gly Ala Val Pro Ala Leu Glu Lys Arg 35 40 45Val Thr Ile Ser Val Ser
Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys 50 55 60Val Gln Val Lys Ile
Gln Asn Pro Thr Ser Cys Thr Ala Ser Gly Thr65 70 75 80Cys Asp Pro
Ser Val Thr Arg Ser Ala Tyr Ala Asp Val Thr Phe Ser 85 90 95Phe Thr
Gln Tyr Ser Thr Asp Glu Glu Arg Ala Leu Val Arg Thr Glu 100 105
110Leu Lys Ala Leu Leu Ala Asp Pro Met Leu Ile Asp Ala Ile Asp Asn
115 120 125Leu Asn Pro Ala Tyr 13014330PRTBacteriophage NL95 14Met
Ala Lys Leu Asn Lys Val Thr Leu Thr Gly Ile Gly Lys Ala Gly1 5 10
15Asn Gln Thr Leu Thr Leu Thr Pro Arg Gly Val Asn Pro Thr Asn Gly
20 25 30Val Ala Ser Leu Ser Glu Ala Gly Ala Val Pro Ala Leu Glu Lys
Arg 35 40 45Val Thr Val Ser Val Ala Gln Pro Ser Arg Asn Arg Lys Asn
Tyr Lys 50 55 60Val Gln Ile Lys Leu Gln Asn Pro Thr Ala Cys Thr Lys
Asp Ala Cys65 70 75 80Asp Pro Ser Val Thr Arg Ser Gly Ser Arg Asp
Val Thr Leu Ser Phe 85 90 95Thr Ser Tyr Ser Thr Glu Arg Glu Arg Ala
Leu Ile Arg Thr Glu Leu 100 105 110Ala Ala Leu Leu Lys Asp Asp Leu
Ile Val Asp Ala Ile Asp Asn Leu 115 120 125Asn Pro Ala Tyr Trp Ala
Ala Leu Leu Ala Ala Ser Pro Gly Gly Gly 130 135 140Asn Asn Pro Tyr
Pro Gly Val Pro Asp Ser Pro Asn Val Lys Pro Pro145 150 155 160Gly
Gly Thr Gly Thr Tyr Arg Cys Pro Phe Ala Cys Tyr Arg Arg Gly 165 170
175Glu Leu Ile Thr Glu Ala Lys Asp Gly Ala Cys Ala Leu Tyr Ala Cys
180 185 190Gly Ser Glu Ala Leu Val Glu Phe Glu Tyr Ala Leu Glu Asp
Phe Leu 195 200 205Gly Asn Glu Phe Trp Arg Asn Trp Asp Gly Arg Leu
Ser Lys Tyr Asp 210 215 220Ile Glu Thr His Arg Arg Cys Arg Gly Asn
Gly Tyr Val Asp Leu Asp225
230 235 240Ala Ser Val Met Gln Ser Asp Glu Tyr Val Leu Ser Gly Ala
Tyr Asp 245 250 255Val Val Lys Met Gln Pro Pro Gly Thr Phe Asp Ser
Pro Arg Tyr Tyr 260 265 270Leu His Leu Met Asp Gly Ile Tyr Val Asp
Leu Ala Glu Val Thr Ala 275 280 285Tyr Arg Ser Tyr Gly Met Val Ile
Gly Phe Trp Thr Asp Ser Lys Ser 290 295 300Pro Gln Leu Pro Thr Asp
Phe Thr Arg Phe Asn Arg His Asn Cys Pro305 310 315 320Val Gln Thr
Val Ile Val Ile Pro Ser Leu 325 33015129PRTBacteriophage f2 15Ala
Ser Asn Phe Thr Gln Phe Val Leu Val Asn Asp Gly Gly Thr Gly1 5 10
15Asn Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu Trp
20 25 30Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser
Val 35 40 45Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val
Glu Val 50 55 60Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu
Pro Val Ala65 70 75 80Ala Trp Arg Ser Tyr Leu Asn Leu Glu Leu Thr
Ile Pro Ile Phe Ala 85 90 95Thr Asn Ser Asp Cys Glu Leu Ile Val Lys
Ala Met Gln Gly Leu Leu 100 105 110Lys Asp Gly Asn Pro Ile Pro Ser
Ala Ile Ala Ala Asn Ser Gly Ile 115 120 125Tyr16128PRTBacteriophage
PP7 16Met Ser Lys Thr Ile Val Leu Ser Val Gly Glu Ala Thr Arg Thr
Leu1 5 10 15Thr Glu Ile Gln Ser Thr Ala Asp Arg Gln Ile Phe Glu Glu
Lys Val 20 25 30Gly Pro Leu Val Gly Arg Leu Arg Leu Thr Ala Ser Leu
Arg Gln Asn 35 40 45Gly Ala Lys Thr Ala Tyr Arg Val Asn Leu Lys Leu
Asp Gln Ala Asp 50 55 60Val Val Asp Cys Ser Thr Ser Val Cys Gly Glu
Leu Pro Lys Val Arg65 70 75 80Tyr Thr Gln Val Trp Ser His Asp Val
Thr Ile Val Ala Asn Ser Thr 85 90 95Glu Ala Ser Arg Lys Ser Leu Tyr
Asp Leu Thr Lys Ser Leu Val Ala 100 105 110Thr Ser Gln Val Glu Asp
Leu Val Val Asn Leu Val Pro Leu Gly Arg 115 120
12517132PRTArtificial SequenceBacteriophage Qbeta 240 mutant 17Ala
Lys Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Arg Asp Gly Lys1 5 10
15Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val
20 25 30Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg
Val 35 40 45Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr
Lys Val 50 55 60Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn
Gly Ser Cys65 70 75 80Asp Pro Ser Val Thr Arg Gln Lys Tyr Ala Asp
Val Thr Phe Ser Phe 85 90 95Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala
Phe Val Arg Thr Glu Leu 100 105 110Ala Ala Leu Leu Ala Ser Pro Leu
Leu Ile Asp Ala Ile Asp Gln Leu 115 120 125Asn Pro Ala Tyr
13018132PRTArtificial SequenceBacteriophage Q-beta 243 mutant 18Ala
Lys Leu Glu Thr Val Thr Leu Gly Lys Ile Gly Lys Asp Gly Lys1 5 10
15Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val
20 25 30Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg
Val 35 40 45Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr
Lys Val 50 55 60Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn
Gly Ser Cys65 70 75 80Asp Pro Ser Val Thr Arg Gln Lys Tyr Ala Asp
Val Thr Phe Ser Phe 85 90 95Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala
Phe Val Arg Thr Glu Leu 100 105 110Ala Ala Leu Leu Ala Ser Pro Leu
Leu Ile Asp Ala Ile Asp Gln Leu 115 120 125Asn Pro Ala Tyr
13019132PRTArtificial SequenceBacteriophage Q-beta 250 mutant 19Ala
Arg Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Arg Asp Gly Lys1 5 10
15Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val
20 25 30Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg
Val 35 40 45Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr
Lys Val 50 55 60Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn
Gly Ser Cys65 70 75 80Asp Pro Ser Val Thr Arg Gln Lys Tyr Ala Asp
Val Thr Phe Ser Phe 85 90 95Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala
Phe Val Arg Thr Glu Leu 100 105 110Ala Ala Leu Leu Ala Ser Pro Leu
Leu Ile Asp Ala Ile Asp Gln Leu 115 120 125Asn Pro Ala Tyr
13020132PRTArtificial SequenceBacteriophage Q-beta 251 mutant 20Ala
Lys Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Lys Asp Gly Arg1 5 10
15Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val
20 25 30Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg
Val 35 40 45Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr
Lys Val 50 55 60Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn
Gly Ser Cys65 70 75 80Asp Pro Ser Val Thr Arg Gln Lys Tyr Ala Asp
Val Thr Phe Ser Phe 85 90 95Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala
Phe Val Arg Thr Glu Leu 100 105 110Ala Ala Leu Leu Ala Ser Pro Leu
Leu Ile Asp Ala Ile Asp Gln Leu 115 120 125Asn Pro Ala Tyr
13021132PRTArtificial SequenceBacteriophage Q-beta 259 mutant 21Ala
Arg Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Lys Asp Gly Arg1 5 10
15Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val
20 25 30Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg
Val 35 40 45Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr
Lys Val 50 55 60Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn
Gly Ser Cys65 70 75 80Asp Pro Ser Val Thr Arg Gln Lys Tyr Ala Asp
Val Thr Phe Ser Phe 85 90 95Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala
Phe Val Arg Thr Glu Leu 100 105 110Ala Ala Leu Leu Ala Ser Pro Leu
Leu Ile Asp Ala Ile Asp Gln Leu 115 120 125Asn Pro Ala Tyr
13022185PRTHepatitis B virus 22Met Asp Ile Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu1 5 10 15Ser Phe Leu Pro Ser Asp Phe Phe
Pro Ser Val Arg Asp Leu Leu Asp 20 25 30Thr Ala Ser Ala Leu Tyr Arg
Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45Ser Pro His His Thr Ala
Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60Leu Met Thr Leu Ala
Thr Trp Val Gly Asn Asn Leu Glu Asp Pro Ala65 70 75 80Ser Arg Asp
Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95Ile Arg
Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105
110Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr
115 120 125Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr
Leu Pro 130 135 140Glu Thr Thr Val Val Arg Arg Arg Asp Arg Gly Arg
Ser Pro Arg Arg145 150 155 160Arg Thr Pro Ser Pro Arg Arg Arg Arg
Ser Gln Ser Pro Arg Arg Arg 165 170 175Arg Ser Gln Ser Arg Glu Ser
Gln Cys 180 18523212PRTHepatitis B virus 23Met Gln Leu Phe His Leu
Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr1 5 10 15Val Gln Ala Ser Lys
Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25 30Asp Pro Tyr Lys
Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45Pro Ser Asp
Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His65 70 75
80His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp Leu Met Asn
85 90 95Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Val Ser Arg
Asp 100 105 110Leu Val Val Gly Tyr Val Asn Thr Thr Val Gly Leu Lys
Phe Arg Gln 115 120 125Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe
Gly Arg Glu Thr Val 130 135 140Ile Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr Pro Pro Ala145 150 155 160Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190Arg Arg
Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195 200
205Glu Ser Gln Cys 21024188PRTHepatitis B virus 24Met Asp Ile Asp
Pro Tyr Lys Glu Phe Gly Ser Ser Tyr Gln Leu Leu1 5 10 15Asn Phe Leu
Pro Leu Asp Phe Phe Pro Asp Leu Asn Ala Leu Val Asp 20 25 30Thr Ala
Thr Ala Leu Tyr Glu Glu Glu Leu Thr Gly Arg Glu His Cys 35 40 45Ser
Pro His His Thr Ala Ile Arg Gln Ala Leu Val Cys Trp Asp Glu 50 55
60Leu Thr Lys Leu Ile Ala Trp Met Ser Ser Asn Ile Thr Ser Glu Gln65
70 75 80Val Arg Thr Ile Ile Val Asn His Val Asn Asp Thr Trp Gly Leu
Lys 85 90 95Val Arg Gln Ser Leu Trp Phe His Leu Ser Cys Leu Thr Phe
Gly Gln 100 105 110His Thr Val Gln Glu Phe Leu Val Ser Phe Gly Val
Trp Ile Arg Thr 115 120 125Pro Ala Pro Tyr Arg Pro Pro Asn Ala Pro
Ile Leu Ser Thr Leu Pro 130 135 140Glu His Thr Val Ile Arg Arg Arg
Gly Gly Ala Arg Ala Ser Arg Ser145 150 155 160Pro Arg Arg Arg Thr
Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro 165 170 175Arg Arg Arg
Arg Ser Gln Ser Pro Ser Thr Asn Cys 180 18525185PRTHepatitis B
virus 25Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu
Leu1 5 10 15Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu
Leu Asp 20 25 30Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro
Glu His Cys 35 40 45Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu
Cys Trp Gly Glu 50 55 60Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn
Leu Glu Asp Pro Ala65 70 75 80Ser Arg Asp Leu Val Val Asn Tyr Val
Asn Thr Asn Met Gly Leu Lys 85 90 95Ile Arg Gln Leu Leu Trp Phe His
Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110Glu Thr Val Leu Glu Tyr
Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125Pro Pro Ala Tyr
Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140Glu Thr
Thr Val Val Arg Arg Arg Asp Arg Gly Arg Ser Pro Arg Arg145 150 155
160Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg
165 170 175Arg Ser Gln Ser Arg Glu Ser Gln Cys 180
18526152PRTHepatitis B virus 26Met Asp Ile Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu1 5 10 15Ser Phe Leu Pro Ser Asp Phe Phe
Pro Ser Val Arg Asp Leu Leu Asp 20 25 30Thr Ala Ala Ala Leu Tyr Arg
Asp Ala Leu Glu Ser Pro Glu His Cys 35 40 45Ser Pro His His Thr Ala
Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp 50 55 60Leu Met Thr Leu Ala
Thr Trp Val Gly Thr Asn Leu Glu Asp Gly Gly65 70 75 80Lys Gly Gly
Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Val 85 90 95Gly Leu
Lys Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr 100 105
110Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp
115 120 125Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile
Leu Ser 130 135 140Thr Leu Pro Glu Thr Thr Val Val145
150273635DNAArtificial Sequenceplasmid pAP283-58 27cgagctcgcc
cctggcttat cgaaattaat acgactcact atagggagac cggaattcga 60gctcgcccgg
ggatcctcta gaattttctg cgcacccatc ccgggtggcg cccaaagtga
120ggaaaatcac atggcaaata agccaatgca accgatcaca tctacagcaa
ataaaattgt 180gtggtcggat ccaactcgtt tatcaactac attttcagca
agtctgttac gccaacgtgt 240taaagttggt atagccgaac tgaataatgt
ttcaggtcaa tatgtatctg tttataagcg 300tcctgcacct aaaccggaag
gttgtgcaga tgcctgtgtc attatgccga atgaaaacca 360atccattcgc
acagtgattt cagggtcagc cgaaaacttg gctaccttaa aagcagaatg
420ggaaactcac aaacgtaacg ttgacacact cttcgcgagc ggcaacgccg
gtttgggttt 480ccttgaccct actgcggcta tcgtatcgtc tgatactact
gcttaagctt gtattctata 540gtgtcaccta aatcgtatgt gtatgataca
taaggttatg tattaattgt agccgcgttc 600taacgacaat atgtacaagc
ctaattgtgt agcatctggc ttactgaagc agaccctatc 660atctctctcg
taaactgccg tcagagtcgg tttggttgga cgaaccttct gagtttctgg
720taacgccgtt ccgcaccccg gaaatggtca ccgaaccaat cagcagggtc
atcgctagcc 780agatcctcta cgccggacgc atcgtggccg gcatcaccgg
cgcacacagt gcggttgctg 840gcgcctatat cgccgacatc accgatgggg
aagatcgggc tcgccacttc gggctcatga 900gcgcttgttt cggcgtgggt
atggtggcag gccccgtggc cgggggactg ttgggcgcca 960tctccttgca
tgcaccattc cttgcggcgg cggtgcttca acggcctcaa cctactactg
1020ggctgcttcc taatgcagga gtcgcataag ggagagcgtc gatatggtgc
actctcagta 1080caatctgctc tgatgccgca tagttaagcc aactccgcta
tcgctacgtg actgggtcat 1140ggctgcgccc cgacacccgc caacacccgc
tgacgcgccc tgacgggctt gtctgctccc 1200ggcatccgct tacagacaag
ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc 1260accgtcatca
ccgaaacgcg cgaggcagct tgaagacgaa agggcctcgt gatacgccta
1320tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
cacttttcgg 1380ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
tacattcaaa tatgtatccg 1440ctcatgagac aataaccctg ataaatgctt
caataatatt gaaaaaggaa gagtatgagt 1500attcaacatt tccgtgtcgc
ccttattccc ttttttgcgg cattttgcct tcctgttttt 1560gctcacccag
aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
1620ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
ccccgaagaa 1680cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
gcgcggtatt atcccgtatt 1740gacgccgggc aagagcaact cggtcgccgc
atacactatt ctcagaatga cttggttgag 1800tactcaccag tcacagaaaa
gcatcttacg gatggcatga cagtaagaga attatgcagt 1860gctgccataa
ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
1920ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
ccttgatcgt 1980tgggaaccgg agctgaatga agccatacca aacgacgagc
gtgacaccac gatgcctgta 2040gcaatggcaa caacgttgcg caaactatta
actggcgaac tacttactct agcttcccgg 2100caacaattaa tagactggat
ggaggcggat aaagttgcag gaccacttct gcgctcggcc 2160cttccggctg
gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
2220atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
ctacacgacg 2280gggagtcagg caactatgga tgaacgaaat agacagatcg
ctgagatagg tgcctcactg 2340attaagcatt ggtaactgtc agaccaagtt
tactcatata tactttagat tgatttaaaa 2400cttcattttt aatttaaaag
gatctaggtg aagatccttt ttgataatct catgaccaaa 2460atcccttaac
gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
2520tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
aaaaccaccg 2580ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
ctctttttcc gaaggtaact 2640ggcttcagca gagcgcagat accaaatact
gtccttctag tgtagccgta gttaggccac 2700cacttcaaga actctgtagc
accgcctaca tacctcgctc tgctaatcct gttaccagtg 2760gctgctgcca
gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
2820gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
cttggagcga 2880acgacctaca
ccgaactgag atacctacag cgcgagcatt gagaaagcgc cacgcttccc
2940gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
agagcgcacg 3000agggagcttc cagggggaaa cgcctggtat ctttatagtc
ctgtcgggtt tcgccacctc 3060tgacttgagc gtcgattttt gtgatgctcg
tcaggggggc ggagcctatg gaaaaacgcc 3120agcaacgcgg cctttttacg
gttcctggcc ttttgctggc cttttgctca catgttcttt 3180cctgcgttat
cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
3240gctcgccgca gccgaacgac gagcgcagcg agtcagtgag cgaggaagcg
gaagagcgcc 3300caatacgcaa accgcctctc cccgcgcgtt ggccgattca
ttaatgcagc tgtggtgtca 3360tggtcggtga tcgccagggt gccgacgcgc
atctcgactg catggtgcac caatgcttct 3420ggcgtcaggc agccatcgga
agctgtggta tggccgtgca ggtcgtaaat cactgcataa 3480ttcgtgtcgc
tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg acatcataac
3540ggttctggca aatattctga aatgagctgt tgacaattaa tcatcgaact
agttaactag 3600tacgcaagtt cacgtaaaaa gggtatcgcg gaatt
363528131PRTBacteriophage AP205 28Met Ala Asn Lys Pro Met Gln Pro
Ile Thr Ser Thr Ala Asn Lys Ile1 5 10 15Val Trp Ser Asp Pro Thr Arg
Leu Ser Thr Thr Phe Ser Ala Ser Leu 20 25 30Leu Arg Gln Arg Val Lys
Val Gly Ile Ala Glu Leu Asn Asn Val Ser 35 40 45Gly Gln Tyr Val Ser
Val Tyr Lys Arg Pro Ala Pro Lys Pro Glu Gly 50 55 60Cys Ala Asp Ala
Cys Val Ile Met Pro Asn Glu Asn Gln Ser Ile Arg65 70 75 80Thr Val
Ile Ser Gly Ser Ala Glu Asn Leu Ala Thr Leu Lys Ala Glu 85 90 95Trp
Glu Thr His Lys Arg Asn Val Asp Thr Leu Phe Ala Ser Gly Asn 100 105
110Ala Gly Leu Gly Phe Leu Asp Pro Thr Ala Ala Ile Val Ser Ser Asp
115 120 125Thr Thr Ala 13029131PRTArtificial SequenceAP205 coat
protein 29Met Ala Asn Lys Thr Met Gln Pro Ile Thr Ser Thr Ala Asn
Lys Ile1 5 10 15Val Trp Ser Asp Pro Thr Arg Leu Ser Thr Thr Phe Ser
Ala Ser Leu 20 25 30Leu Arg Gln Arg Val Lys Val Gly Ile Ala Glu Leu
Asn Asn Val Ser 35 40 45Gly Gln Tyr Val Ser Val Tyr Lys Arg Pro Ala
Pro Lys Pro Glu Gly 50 55 60Cys Ala Asp Ala Cys Val Ile Met Pro Asn
Glu Asn Gln Ser Ile Arg65 70 75 80Thr Val Ile Ser Gly Ser Ala Glu
Asn Leu Ala Thr Leu Lys Ala Glu 85 90 95Trp Glu Thr His Lys Arg Asn
Val Asp Thr Leu Phe Ala Ser Gly Asn 100 105 110Ala Gly Leu Gly Phe
Leu Asp Pro Thr Ala Ala Ile Val Ser Ser Asp 115 120 125Thr Thr Ala
130303607DNAArtificial Sequenceplasmid pAP281-32 30cgagctcgcc
cctggcttat cgaaattaat acgactcact atagggagac cggaattcga 60gctcgcccgg
ggatcctcta gattaaccca acgcgtagga gtcaggccat ggcaaataag
120acaatgcaac cgatcacatc tacagcaaat aaaattgtgt ggtcggatcc
aactcgttta 180tcaactacat tttcagcaag tctgttacgc caacgtgtta
aagttggtat agccgaactg 240aataatgttt caggtcaata tgtatctgtt
tataagcgtc ctgcacctaa accgaaggtc 300agatgcctgt gtcattatgc
cgaatgaaaa ccaatccatt cgcacagtga tttcagggtc 360agccgaaaac
ttggctacct taaaagcaga atgggaaact cacaaacgta acgttgacac
420actcttcgcg agcggcaacg ccggtttggg tttccttgac cctactgcgg
ctatcgtatc 480gtctgatact actgcttaag cttgtattct atagtgtcac
ctaaatcgta tgtgtatgat 540acataaggtt atgtattaat ggtagccgcg
ttctaacgac aatatgtaca agcctaattg 600tgtagcatct ggcttactga
agcagaccct atcatctctc tcgtaaactg ccgtcagagt 660cggttgggtt
ggacagacct ctgagtttct ggtaacgccg ttccgcaccc cggaaatggt
720caccgaacca ttcagcaggg tcatcgctag ccagatcctc tacgccggac
gcatcgtggc 780ccgcatcacc ggcgccacag gtgcggtgct ggcgcctata
tcgccgacat caccgatggg 840gaagatcggg ctcgccactt cgggctcatg
atcgctggtt tccgcctggg tatggtggca 900ggccccgtgg cccgggggac
tgttgggcgc catctccttg catgcaccat tccttgcggc 960ggcggtgctc
aacggcctca acctactact gggctgcttc ctaatgcagg agtcgcataa
1020gggagagcgt cgatatggtg cactctcagt acaatctgct ctgatgccgc
atagttaagc 1080caactccgct atcgctacgt gactgggtca tggctgcgcc
ccgacacccg ccaacacccg 1140ctgacgcgcc ctgacgggct tgtctgcttc
cggcatccgc ttacagacaa gctgtgaccg 1200tctccgggag ctgcatgtgt
cagaggtttt caccgtcatc accgaaacgc gcgaggcagc 1260ttgaagacga
aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
1320ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggacccc
ctattggttt 1380atttttctaa atacattcaa atatgtatcc gctcatgaga
caataaccct gataaatgct 1440tcaataatat tgaaaaagga agagtatgag
tattcaacat ttccgtgtcg cccttattcc 1500cttttttgcg gcattttgcc
ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 1560agatgctgaa
gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
1620taagatcctt gagagttttc gccccgaaga acgtttttca atgatgagca
cttttaaagt 1680tctgctatgt gtcgcggtat tatcccgtat tgacgccggg
caagagcaac tcggtcgccg 1740catacactat tctcagaatg acttggtggt
acctaccagt cacagaaaag catcttacgg 1800atggcatgac agtaagagaa
ttatgcagtg ctgccataac catgagtgat aacactgcgg 1860ccaacttact
tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
1920tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
gccataccaa 1980acgacgagcg tgacaccacg atgcctgtac gaacggcaac
aacgttgcgc aaactattaa 2040ctggcgaact acttactcta gcttcccggc
aacaattaat agactggatg gaggcggata 2100aagttgcagg accacttctg
cgctcggccc ttccggctgg ctggtttatt gctgataaat 2160ctggagccgg
tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
2220cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
gaacgaaata 2280gacagatcgc tgagataggt gcctcactga ttaagcattg
gtaactgtca gaccaagttt 2340actcatatat actttagatt gatttaaaac
ttcattttta atttaaaagg atctaggtga 2400agatcctttt tgataatctc
atgaccaaaa tcccttaacg tgagttttcg ttccactgag 2460cggtcagacc
ccgtagaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
2520tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
ccggatcaag 2580agctaccaac tctttttccg aaggtaactg gcttcagcag
agcgcagata ccaaatactg 2640tccttctagt gtagccgtag ttaggccacc
acttcaagaa ctctgtagca ccgcctacat 2700acctcgctct gctaatcctg
ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 2760ccgggttgga
ctcaagacga taggtaccgg ataaggcgca gcggtcgggc tgaacggggg
2820gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
tacctacagc 2880gcgagcattg agaaagcgcc acgcttcccg aagggagaaa
ggcggacagg tatccggtaa 2940gcggcagggt cggaacaaga gagcgcacga
gggagcttcc agggggaaac gcctggtatc 3000tttatagtcc tgtcgggttt
cgccacctct gacttgagcg tcgatttttg tgatgctcgt 3060caggggggcg
gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
3120ttggctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
gtggataacc 3180gtattaccgc ctttgagtga gctgataccg ctcgccgcag
ccgaacgacc gacggcgcag 3240cgagtcagtg agcgaggaag cggaagagcg
cccaatacgc aaaccgcctc tccccgcgcg 3300ttggccgatt cattaatgca
gctgtggtgt catggtcggt gatcgccagg gtgccgacgc 3360gcatctcgac
tgcatggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg
3420tatggccgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg
cactcccgtt 3480ctggataatg ttttttgcgg cgacatcata acggttctgg
caaatattct gaaatgagct 3540ggtgacaatt aatcatcgaa ctagttaact
agtacgcaag ttcacgtaaa aagggtatcg 3600cggaatt 3607316PRTArtificial
SequenceN-terminal linker 31Cys Gly Asp Glu Gly Gly1
5326PRTArtificial SequenceC-terminal linker 32Gly Gly Glu Asp Gly
Cys1 5335PRTArtificial SequenceLinker 33Gly Gly Lys Gly Gly1
5343PRTArtificial SequenceN-terminal glycine linker 34Gly Cys
Gly1359PRTArtificial SequenceN terminal glycine serine linkers
35Gly Cys Gly Ser Gly Gly Gly Gly Ser1 5363PRTArtificial
SequenceC-terminal glycine linker 36Gly Cys Gly13710PRTArtificial
SequenceC terminal glycine serine linkers 37Gly Ser Gly Gly Gly Gly
Ser Gly Cys Gly1 5 10385PRTArtificial SequenceGlycine serine linker
38Gly Gly Gly Gly Ser1 53910PRTArtificial SequenceN-terminal gamma1
39Cys Gly Asp Lys Thr His Thr Ser Pro Pro1 5 104010PRTArtificial
SequenceC-terminal gamma 1 40Asp Lys Thr His Thr Ser Pro Pro Cys
Gly1 5 104117PRTArtificial SequenceN-terminal gamma 3 41Cys Gly Gly
Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser Gly Gly Ala1 5 10
15Pro4218PRTArtificial SequenceC-terminal gamma 3 42Pro Lys Pro Ser
Thr Pro Pro Gly Ser Ser Gly Gly Ala Pro Gly Gly1 5 10 15Cys
Gly436PRTArtificial SequenceN-terminal glycine linker 43Gly Cys Gly
Gly Gly Gly1 5446PRTArtificial SequenceC-terminal glycine linker
44Gly Gly Gly Gly Cys Gly1 5456PRTArtificial SequenceC-terminal
glycine-lysine linker 45Gly Gly Lys Lys Gly Cys1 5466PRTArtificial
SequenceN-terminal glycine-lysine linker 46Cys Gly Lys Lys Gly Gly1
5474PRTArtificial SequenceC.terminal linker 47Gly Gly Cys
Gly14837DNAArtificial SequenceOligonucleotide primer 48ggtaacatcg
gtcgagatgg aaaacaaact ctggtcc 374937DNAArtificial
SequenceOligonucleotide primer 49ggaccagagt ttgttttcca tctcgaccga
tgttacc 375022DNAArtificial SequenceOligonucleotide primer
50agctcgcccg gggatcctct ag 225140DNAArtificial
SequenceOligonucleotide primer 51cgatgcattt catccttagt tatcaatacg
ctgggttcag 405236DNAArtificial SequenceOligonucleotide primer
52ggcaaaatta gagactgtta ctttaggtaa gatcgg 365336DNAArtificial
SequenceOligonucleotide primer 53ccgatcttac ctaaagtaac agtctctaat
tttgcc 365433DNAArtificial SequenceOligonucleotide primer
54ggccatggca cgactcgaga ctgttacttt agg 335519DNAArtificial
SequenceOligonucleotide primer 55gatttaggtg acactatag
195637DNAArtificial SequenceOligonucleotide primer 56gatggacgtc
aaactctggt cctcaatccg cgtgggg 375737DNAArtificial
SequenceOligonucleotide primer 57ccccacgcgg attgaggacc agagtttgac
gtccatc 375831DNAArtificial SequenceEcoRIHBcAg(s) primer
58ccggaattca tggacattga cccttataaa g 315951DNAArtificial
SequenceLys-HBcAg(as) primer 59cctagagcca cctttgccac catcttctaa
attagtaccc acccaggtag c 516048DNAArtificial SequenceLys-HBcAg(s)
primer 60gaagatggtg gcaaaggtgg ctctagggac ctagtagtca gttatgtc
486138DNAArtificial SequenceHBcAg(1-149)Hind(as) primer
61cgcgtcccaa gcttctaaac aacagtagtc tccggaag 386237DNAArtificial
Sequence48as primer 62gtgcagtatg gtgaggtgag gaatgctcag gagactc
376337DNAArtificial Sequence48s primer 63gagtctcctg agcattcctc
acctcaccat actgcac 376433DNAArtificial Sequence107as primer
64cttccaaaag tgagggaaga aatgtgaaac cac 336547DNAArtificial
SequenceHBcAg149hind-as 65cgcgtcccaa gcttctaaac aacagtagtc
tccggaagcg ttgatag 476633DNAArtificial Sequence107s primer
66gtggtttcac atttcttccc tcacttttgg aag 336738DNAArtificial
SequenceHBcAgwtHindIIII primer 67cgcgtcccaa gcttctaaca ttgagattcc
cgagattg 386810PRTArtificial Sequenceepitope CeH3 68Val Asn Leu Thr
Trp Ser Arg Ala Ser Gly1 5 106951DNAArtificial SequenceCeH3fwd
primer 69gtt aac ttg acc tgg tct cgt gct tct ggt gca tcc agg gat
cta gta 48Val Asn Leu Thr Trp Ser Arg Ala Ser Gly Ala Ser Arg Asp
Leu Val1 5 10 15gtc 51Val7017PRTArtificial SequenceCeH3fwd primer
70Val Asn Leu Thr Trp Ser Arg Ala Ser Gly Ala Ser Arg Asp Leu Val1
5 10 15Val7151DNAArtificial SequenceCeH3rev primer 71accagaagca
cgagaccagg tcaagttaac atcttccaaa ttattaccca c 51727PRTArtificial
SequenceCeH3rev primer peptide 72Asp Glu Leu Asn Asn Gly Val1
57331DNAArtificial SequenceHBcAg-wt EcoRI fwd primer 73ccggaattca
tggacattga cccttataaa g 317438DNAArtificial SequenceHBcAg-wt Hind
III rev primer 74cgcgtcccaa gcttctaaca ttgagattcc cgagattg
38756PRTHomo sapiens 75Asp Ala Glu Phe Arg His1 5766PRTMus musculus
76Asp Ala Glu Phe Gly His1 5779PRTArtificial SequenceAbeta 1-6 GGC
77Asp Ala Glu Phe Arg His Gly Gly Cys1 5789PRTArtificial
Sequencemurine Abeta 1-6 GGC 78Asp Ala Glu Phe Gly His Gly Gly Cys1
57928DNAArtificial Sequenceprimer p1.44 79aaccatggca aataagccaa
tgcaaccg 288030DNAArtificial Sequenceprimer p1.45 80aatctagaat
tttctgcgca cccatcccgg 308130DNAArtificial Sequenceprimer p1.46
81aaaagcttaa gcagtagtat cagacgatac 308243DNAArtificial
Sequenceprimer p1.47 82gagtgatcca actcgtttat caactacatt ttcagcaagt
ctg 438343DNAArtificial Sequenceprimer p1.48 83cagacttgct
gaaaatgtag ttgataaacg agttggatca ctc 43846PRThomo sapiens 84Asp Ala
Glu Phe Arg His1 5856PRTOryctolagus cuniculus 85Asp Ala Glu Phe Arg
His1 5866PRTXenopus laevis 86Asp Ser Glu Tyr Arg His1 5876PRTRattus
norvegicus 87Asp Ala Glu Phe Gly His1 5886PRTCavia porcellus 88Asp
Ala Glu Phe Arg His1 58915PRTMus musculus 89Val His Glu Pro His Glu
Phe Arg His Val Ala Leu Asn Pro Val1 5 10 15906PRTMus musculus
90Tyr Tyr Glu Phe Arg His1 59142PRTHomo sapiens 91Asp Ala Glu Phe
Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe
Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu
Met Val Gly Gly Val Val Ile Ala 35 4092770PRTHomo sapiens 92Met Leu
Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg1 5 10 15Ala
Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25
30Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln
35 40 45Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile
Asp 50 55 60Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro
Glu Leu65 70 75 80Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val
Thr Ile Gln Asn 85 90 95Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr
His Pro His Phe Val 100 105 110Ile Pro Tyr Arg Cys Leu Val Gly Glu
Phe Val Ser Asp Ala Leu Leu 115 120 125Val Pro Asp Lys Cys Lys Phe
Leu His Gln Glu Arg Met Asp Val Cys 130 135 140Glu Thr His Leu His
Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu145 150 155 160Lys Ser
Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 165 170
175Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu
180 185 190Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser
Asp Val 195 200 205Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly
Ser Glu Asp Lys 210 215 220Val Val Glu Val Ala Glu Glu Glu Glu Val
Ala Glu Val Glu Glu Glu225 230 235 240Glu Ala Asp Asp Asp Glu Asp
Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255Glu Ala Glu Glu Pro
Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270Ala Thr Thr
Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285Glu
Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile 290 295
300Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe
Phe305 310 315 320Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp
Thr Glu Glu Tyr 325 330 335Cys Met Ala Val Cys Gly Ser Ala Met Ser
Gln Ser Leu Leu Lys Thr 340 345 350Thr Gln Glu Pro Leu Ala Arg Asp
Pro Val Lys Leu Pro Thr Thr Ala 355 360 365Ala Ser Thr Pro Asp Ala
Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp 370 375 380Glu Asn Glu His
Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala385 390 395 400Lys
His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala 405 410
415Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile
420 425 430Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala
Ala Asn 435 440 445Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg
Val Glu Ala Met 450 455 460Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu
Asn Tyr Ile Thr Ala Leu465 470
475 480Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys
Lys 485 490 495Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu
Lys His Phe 500 505 510Glu His Val Arg Met Val Asp Pro Lys Lys Ala
Ala Gln Ile Arg Ser 515 520 525Gln Val Met Thr His Leu Arg Val Ile
Tyr Glu Arg Met Asn Gln Ser 530 535 540Leu Ser Leu Leu Tyr Asn Val
Pro Ala Val Ala Glu Glu Ile Gln Asp545 550 555 560Glu Val Asp Glu
Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val 565 570 575Leu Ala
Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala 580 585
590Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro
595 600 605Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His
Ser Phe 610 615 620Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu
Val Glu Pro Val625 630 635 640Asp Ala Arg Pro Ala Ala Asp Arg Gly
Leu Thr Thr Arg Pro Gly Ser 645 650 655Gly Leu Thr Asn Ile Lys Thr
Glu Glu Ile Ser Glu Val Lys Met Asp 660 665 670Ala Glu Phe Arg His
Asp Ser Gly Tyr Glu Val His His Gln Lys Leu 675 680 685Val Phe Phe
Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly 690 695 700Leu
Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu705 710
715 720Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val
Val 725 730 735Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu
Ser Lys Met 740 745 750Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys
Phe Phe Glu Gln Met 755 760 765Gln Asn 7709382PRTHomo sapiens 93Gly
Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys1 5 10
15Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln
20 25 30Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala
Ile 35 40 45Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile
Ile Ile 50 55 60Thr Leu Val Met Leu Lys Lys Gln Tyr Thr Ser Asn His
His Gly Val65 70 75 80Val Glu
* * * * *